Certain chemical entities, compositions and methods

ABSTRACT

Chemical entities that modulate PI3 kinase activity, and chemical entities, pharmaceutical compositions, and methods of treatments of diseases and conditions associated with P13 kinase activity are described herein.

This application is a divisional application of U.S. application Ser.No. 15/059,962, filed Mar. 3, 2016, which is a divisional application ofU.S. application Ser. No. 14/222,488, filed Mar. 21, 2014, nowabandoned, which is a continuation application of U.S. application Ser.No. 12/811,695, now U.S. Pat. No. 8,703,777, which is a national phaseentry pursuant to 35 U.S.C. § 371 of International Patent ApplicationNo. PCT/US2009/000042, filed on Jan. 5, 2009, which claims the benefitof U.S. Provisional Application Ser. No. 61/009,971, filed on Jan. 4,2008, 61/194,294, filed on Sep. 26, 2008, and 61/201,146, filed on Dec.5, 2008, each of which is hereby incorporated by reference in itsentirety.

SEQUENCE LISTING

The present application is being filed with a Sequence Listing submittedas filename 12928-155-999_SeqListing.txt, of size 973 bytes, which wascreated on May 14, 2016. The Sequence Listing is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

The activity of cells can be regulated by external signals thatstimulate or inhibit intracellular events. The process by whichstimulatory or inhibitory signals are transmitted into and within a cellto elicit an intracellular response is referred to as signaltransduction. Over the past decades, cascades of signal transductionevents have been elucidated and found to play a central role in avariety of biological responses. Defects in various components of signaltransduction pathways have been found to account for a vast number ofdiseases, including numerous forms of cancer, inflammatory disorders,metabolic disorders, vascular and neuronal diseases (Gaestel et al.Current Medicinal Chemistry (2007) 14:2214-2234).

Kinases represent a class of important signaling molecules. Kinases cangenerally be classified into protein kinases and lipid kinases, andcertain kinases exhibit dual specificities. Protein kinases are enzymesthat phosphorylate other proteins and/or themselves (i.e.,autophosphorylation). Protein kinases can be generally classified intothree major groups based upon their substrate utilization: tyrosinekinases which predominantly phosphorylate substrates on tyrosineresidues (e.g., erb2, PDGF receptor, EGF receptor, VEGF receptor, src,abl), serine/threonine kinases which predominantly phosphorylatesubstrates on serine and/or threonine residues (e.g., mTorC1, mTorC2,ATM, ATR, DNA-PK, Akt), and dual-specificity kinases which phosphorylatesubstrates on tyrosine, serine and/or threonine residues.

Lipid kinases are enzymes that catalyze the phosphorylation of lipids.These enzymes, and the resulting phosphorylated lipids and lipid-derivedbiologically active organic molecules, play a role in many differentphysiological processes, including cell proliferation, migration,adhesion, and differentiation. Certain lipid kinases are membraneassociated and they catalyze the phosphorylation of lipids contained inor associated with cell membranes. Examples of such enzymes includephosphoinositide(s) kinases (such as PI3-kinases, PI4-Kinases),diacylglycerol kinases, and sphingosine kinases.

The phosphoinositide 3-kinases (PI3Ks) signaling pathway is one of themost highly mutated systems in human cancers. PI3K signaling is also akey factor in many other diseases in humans. PI3K signaling is involvedin many disease states including allergic contact dermatitis, rheumatoidarthritis, osteoarthritis, inflammatory bowel diseases, chronicobstructive pulmonary disorder, psoriasis, multiple sclerosis, asthma,disorders related to diabetic complications, and inflammatorycomplications of the cardiovascular system such as acute coronarysyndrome.

PI3Ks are members of a unique and conserved family of intracellularlipid kinases that phosphorylate the 3′-OH group onphosphatidylinositols or phosphoinositides. The PI3K family comprises 15kinases with distinct substrate specificities, expression patterns, andmodes of regulation (Katso et al., 2001). The class I PI3Ks (p110α,p110β, p110δ, and p110γ) are typically activated by tyrosine kinases orG-protein coupled receptors to generate PIP3, which engages downstreameffectors such as those in the Akt/PDK1 pathway, mTOR, the Tec familykinases, and the Rho family GTPases. The class II and III PI3-Ks play akey role in intracellular trafficking through the synthesis of PI(3)Pand PI(3,4)P2. The PIKKs are protein kinases that control cell growth(mTORC1) or monitor genomic integrity (ATM, ATR, DNA-PK, and hSmg-1).

The delta (δ) isoform of class I PI3K has been implicated, inparticular, in a number of diseases and biological processes. PI3K δ isexpressed primarily in hematopoietic cells including leukocytes such asT-cells, dendritic cells, neutrophils, mast cells, B-cells, andmacrophages. PI3K δ is integrally involved in mammalian immune systemfunctions such as T-cell function, B-cell activation, mast cellactivation, dendritic cell function, and neutrophil activity. Due to itsintegral role in immune system function, PI3K δ is also involved in anumber of diseases related to undesirable immune response such asallergic reactions, inflammatory diseases, inflammation mediatedangiogenesis, rheumatoid arthritis, auto-immune diseases such as lupus,asthma, emphysema and other respiratory diseases. Other class I PI3Kinvolved in immune system function includes PI3K γ, which plays a rolein leukocyte signaling and has been implicated in inflammation,rheumatoid arthritis, and autoimmune diseases such as lupus.

Downstream mediators of the PI3K signal transduction pathway include Aktand mammalian target of rapamycin (mTOR). Akt possesses a plckstrinhomology (PH) domain that binds PIP3, leading to Akt kinase activation.Akt phosphorylates many substrates and is a central downstream effectorof PI3K for diverse cellular responses. One important function of Akt isto augment the activity of mTOR, through phosphorylation of TSC2 andother mechanisms. mTOR is a serine-threonine kinase related to the lipidkinases of the PI3K family. mTOR has been implicated in a wide range ofbiological processes including cell growth, cell proliferation, cellmotility and survival. Disregulation of the mTOR pathway has beenreported in various types of cancer. mTOR is a multifunctional kinasethat integrates growth factor and nutrient signals to regulate proteintranslation, nutrient uptake, autophagy, and mitochondrial function.

As such, kinases, particularly PI3Ks are prime targets for drugdevelopment. There remains a need for PI3K inhibitors suitable for drugdevelopment. The present invention addresses this need and providesrelated advantages as well by providing new classes of kinaseinhibitors.

SUMMARY OF THE INVENTION

In a first aspect of the invention, compounds are provided which are ofFormula I, or their pharmaceutically acceptable salts thereof, wherein

W_(a) ¹ is CR³ or N; W_(a) ² is CR⁵ or N; W_(a) ³ is CR⁶ or N; W_(a) ⁴is N or CR⁷; W_(b) ⁵ is CR⁸, CHR⁸, or N, wherein no more than twoadjacent ring atoms selected from W_(a) ¹, W_(a) ², W_(a) ³, W_(a) ⁴,and W_(b) ⁵ are heteroatoms. W_(d) is heterocycloalkyl, aryl orheteroaryl. B is alkyl, amino, heteroalkyl, cycloalkyl,heterocycloalkyl, or a moiety of Formula II;

wherein W_(c) is aryl, heteroaryl, heterocycloalkyl, or cycloalkyl, andq is an integer of 0, 1, 2, 3, or 4. X is absent or is —(CH(R⁹))_(z)—and each instance of z is independently an integer of 1, 2, 3, or 4. Yis absent, —O—, —S—, —S(═O)—, —S(═O)₂—, —N(R⁹)—, —C(═O)—(CHR⁹)_(z)—,—C(═O)—, —N(R⁹)—C(═O)—, or —N(R⁹)—C(═O)NH—, —N(R⁹)C(R⁹)₂—, or—C(═O)—(CHR⁹)_(z)—; wherein when W_(b) ⁵ is N, no more than one of X orY is absent. R¹ is hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl,sulfonamido, halo, cyano, hydroxy, nitro, phosphate, urea, or carbonate.R² is alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, alkoxy,amido, amino, acyl, acyloxy, alkoxycarbonyl, sulfonamido, halo, cyano,hydroxy, nitro, phosphate, urea, or carbonate. R³ is hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkoxy, amido, amino,acyl, acyloxy, sulfonamido, halo, cyano, hydroxy or nitro. R⁵, R⁶, R⁷,and R⁸ are independently hydrogen, C₁-C₄alkyl, C₂-C₅alkenyl,C₂-C₅alkynyl, C₃-C₅cycloalkyl, heterocycloalkyl, C₁-C₄heteroalkyl,C₁-C₄alkoxy, C₁-C₄amido, amino, acyl, C₁-C₄acyloxy, C₁-C₄sulfonamido,halo, cyano, hydroxy or nitro. Each instance of R⁹ is independentlyhydrogen, C₁-C₁₀alkyl, C₃-C₇cycloalkyl, or C₂-C₁₀heteroalkyl.

In another aspect of the invention, compounds are provided which are ofFormula IX or their pharmaceutically acceptable salts thereof, wherein

-   -   W_(a) ¹ and W_(a) ² are independently CR⁵, S, N, or NR⁴, and        W_(a) ⁴ is independently CR⁷, S, N, or NR⁴ wherein no more than        two adjacent ring atoms are nitrogen or sulfur, and when W_(a) ¹        is S, one of W_(a) ² and W_(a) ⁴ is N or NR⁴. W_(b) ⁵ is CR⁸, N,        or NR⁸. B is alkyl, amino, heteroalkyl, cycloalkyl,        heterocycloalkyl, or a moiety of Formula II:

-   -   wherein W_(c) is aryl, heteroaryl, heterocycloalkyl, or        cycloalkyl, and q is an integer of 0, 1, 2, 3, or 4. W_(d) is        absent or is a heterocycloalkyl, aryl or heteroaryl moiety. X is        absent or is —(CH(R⁹))_(z)— and each instance of z independently        is an integer of 1, 2, 3, or 4. Y is absent, —O—, —S—, —S(═O)—,        —S(═O)2-, —N(R9)-, —C(═O)—(CHR9)z-, —C(═O)—, —N(R9)-C(═O)—, or        —N(R9)-C(═O)NH—, —N(R9)C(R9)2-, or —C(═O)—(CHR9)z-. R¹ is        hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,        heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl,        alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl,        sulfonamido, halo, cyano, hydroxy, nitro, phosphate, urea, or        carbonate. R² is alkyl, heteroalkyl, alkenyl, alkynyl,        cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,        heteroarylalkyl, alkoxy, amido, amino, acyl, acyloxy,        alkoxycarbonyl, sulfonamido, halo, cyano, hydroxy, nitro,        phosphate, urea, or carbonate. R³ is hydrogen, alkyl, alkenyl,        alkynyl, cycloalkyl, heterocycloalkyl, alkoxy, amido, amino,        acyl, acyloxy, sulfonamido, halo, cyano, hydroxy or nitro. R⁴ is        hydrogen, acyl, C₁-C₄alkyl, C₂-C₅alkenyl, C₂-C₅alkynyl,        C₃-C₅cycloalkyl, or C₁-C₄heteroalkyl; R⁵, R⁷, and R⁸ are        independently hydrogen, C₁-C₄alkyl, C₂-C₅alkenyl, C₂-C₅alkynyl,        C₃-C₅cycloalkyl, C₁-C₄heteroalkyl, acyl, C₁-C₄alkoxy,        C₁-C₄amido, amino, C₁-C₄acyloxy, C₁-C₄sulfonamido, halo, cyano,        hydroxy or nitro. Each instance of R⁹ is independently hydrogen,        C₁-C₁₀alkyl, C₃-C₇cycloalkyl, or C₂-C₁₀heteroalkyl.

In some embodiments, the compound of Formula I has a structure ofFormula IV:

In some embodiments a compound of Formula IV is of Formula V or VI:

In some embodiments, the compound of Formula VI has the structure ofFormula VI-A:

In some embodiments of the compound of Formula VI-A, R¹¹ is amino. Insome embodiments of the compound of Formula VI-A, R¹² is alkyl, alkenyl,alkynyl, heteroaryl, aryl, heterocycloalkyl, cyano, amino, carboxylicacid, or amido. In some embodiments of the compound of Formula VI-A, R¹²is a monocyclic heteroaryl or a bicyclic heteroaryl.

In some embodiments of the compound of Formula VI, the compound has thestructure of Formula VI-C:

In some of the embodiments of Formula VI, the compound has a structureof Formula VI-D;

In another aspect of the invention a compound or its pharmaceuticallyacceptable salts having the structure of Formula VI is provided,wherein: B is alkyl, amino, heteroalkyl, or a moiety of Formula II;wherein W_(c) is aryl, heteroaryl, heterocycloalkyl, or cycloalkyl, andq is an integer of 0, 1, 2, 3, or 4; X is absent or —(CH(R⁹))_(z)— and zis an integer of 1, 2, 3, or 4; Y is absent, —N(R⁹)—, or —N(R⁹)—CH(R⁹)—;W_(d) is:

R¹ is hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, alkoxy,amido, amino, acyl, acyloxy, alkoxycarbonyl, sulfonamido, halo, cyano,hydroxy, nitro, phosphate, urea, or carbonate; R² is alkyl, heteroalkyl,alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl,heteroaryl, heteroarylalkyl, alkoxy, amido, amino, acyl, acyloxy,alkoxycarbonyl, sulfonamido, halo, cyano, hydroxy, nitro, or phosphate;

R³ is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl, sulfonamido, halo,cyano, hydroxy, nitro, aryl or heteroaryl; each instance of R⁹ isindependently hydrogen, C₁-C₁₀alkyl, C₃-C₇cycloalkyl, heterocycloalkyl,or C₂-C₁₀heteroalkyl; and R¹² is H, alkyl, alkynyl, alkenyl, halo, aryl,heteroaryl, heterocycloalkyl, cycloalkyl, cyano, amino, carboxylic acid,alkoxycarbonyl or amido.

In some embodiments, the compound of Formula VI has the structure ofFormula 6-A:

In some embodiments, the compound of Formula VI has the structure ofFormula 6-C1:

In some embodiments, the compound of Formula VI has the structure ofFormula 6-C2:

In some embodiments, the compound of Formula VI has the structure ofFormula 6-D:

In some embodiments a compound of Formula I is of Formula VII:

In some embodiments, the compound of Formula I has a structure ofFormula VIII:

-   -   where X is absent and Y is —O—, —S—, —S(═O)—, —S(═O)₂—, —N(R⁹)—,        —C(═O)—(CHR⁹)_(z)—, —C(═O)—, —N(R⁹)(C═O)—, or —N(R⁹)(C═O)NH—; or        X is —(CH(R⁹))_(z)—, and Y is absent; or X is —(CH(R⁹))_(z)—,        and Y is O—, —S—, —S(═O)—, —S(═O)₂—, —N(R⁹)—,        —C(═O)—(CHR⁹)_(z)—, —C(═O)—, —N(R⁹)—C(═O)—, or —N(R⁹)—C(═O)NH—,        —N(R⁹)C(R⁹)₂—, or —C(═O)—(CHR⁹)_(z). Each instance of z        independently is an integer of 1, 2, 3, or 4. W_(d) is bicyclic        aryl or bicyclic heteroaryl.

In some embodiments of the compound of Formula IX, the compound has astructure which is a member of the group consisting of: (i) W_(a) ¹ isNR⁴, W_(a) ² is CR⁵, W_(a) ⁴ is CR⁷, and W_(b) ⁵ is CR⁸; (ii) W_(a) ¹ isNR⁴, W_(a) ² is CR⁵, W_(a) ⁴ is CR⁷, and W_(b) ⁵ is CHR⁸; (iii) W_(a) ¹is NR⁴, W_(a) ² is CR⁵, W_(a) ⁴ is CR⁷, and W_(b) ⁵ is N; (iv) W_(a) ¹is NR⁴, W_(a) ² is CR⁵, W_(a) ⁴ is CR⁷, and W_(b) ⁵ is NR⁸; (v) W_(a) ¹is NR⁴, W_(a) ² is N, W_(a) ⁴ is CR⁷, and W_(b) ⁵ is CR⁸; (vi) W_(a) ¹is NR⁴, W_(a) ² is N, W_(a) ⁴ is CR⁷, and W_(b) ⁵ is CHR⁸; (vii) W_(a) ¹is NR⁴, W_(a) ² is N, W_(a) ⁴ is CR⁷, and W_(b) ⁵ is N; (viii) W_(a) ¹is NR⁴, W_(a) ² is N, W_(a) ⁴ is CR⁷, and W_(b) ⁵ is NR⁸; (ix) W_(a) ¹is NR⁴, W_(a) ² is CR⁵, W_(a) ⁴ is N, and W_(b) ⁵ is CR⁸; (x) W_(a) ¹ isNR⁴, W_(a) ² is CR⁵, W_(a) ⁴ is N, and W_(b) ⁵ is CHR⁸; (xi) W_(a) ¹ isNR⁴, W_(a) ² is CR⁵, W_(a) ⁴ is N, and W_(b) ⁵ is N; (xii) W_(a) ¹ isNR⁴, W_(a) ² is CR⁵, W_(a) ⁴ is N, and W_(b) ⁵ is NR⁸; (xiii) W_(a) ¹ isS, W_(a) ² is CR⁵, W_(a) ⁴ is N, and W_(b) ⁵ is CR⁸; (xiv) W_(a) ¹ is S,W_(a) ² is CR⁵, W_(a) ⁴ is N, and W_(b) ⁵ is CHR⁸; (xv) W_(a) ¹ is S,W_(a) ² is CR⁵, W_(a) ⁴ is N, and W_(b) ⁵ is N; (xvi) W_(a) ¹ is S,W_(a) ² is CR⁵, W_(a) ⁴ is N, and W_(b) ⁵ is NR⁸; (xvii) W_(a) ¹ is N,W_(a) ² is CR⁵, W_(a) ⁴ is S, and W_(b) ⁵ is CR⁸; (xviii) W_(a) ¹ is N,W_(a) ² is CR⁵, W_(a) ⁴ is S, and W_(b) ⁵ is CHR⁸; (xix) W_(a) ¹ is N,W_(a) ² is CR⁵, W_(a) ⁴ is S, and W_(b) ⁵ is N; (xx) W_(a) ¹ is N, W_(a)² is CR⁵, W_(a) ⁴ is S, and W_(b) ⁵ is NR⁸; (xxi) W_(a) ¹ is CR⁵, W_(a)² is N, W_(a) ⁴ is S, and W_(b) ⁵ is CR⁸; (xxi) W_(a) ¹ is CR⁵, W_(a) ²is N, W_(a) ⁴ is S, and W_(b) ⁵ is CHR⁸; (xxii) W_(a) ¹ is CR⁵, W_(a) ²is N, W_(a) ⁴ is S, and W_(b) ⁵ is N; (xxiii) W_(a) ¹ is CR⁵, W_(a) ² isN, W_(a) ⁴ is S, and W_(b) ⁵ is NR⁸; (xxiv) W_(a) ¹ is S, W_(a) ² is N,W_(a) ⁴ is CR⁷, and W_(b) ⁵ is CR⁸; (xxv) W_(a) ¹ is S, W_(a) ² is N,W_(a) ⁴ is CR⁷, and W_(b) ⁵ is CHR⁸; (xxvi) W_(a) ¹ is S, W_(a) ² is N,W_(a) ⁴ is CR⁷, and W_(b) ⁵ is N; (xxvii) W_(a) ¹ is S, W_(a) ² is N,W_(a) ⁴ is CR⁷, and W_(b) ⁵ is NR⁸; (xxviii) W_(a) ¹ is CR⁵, W_(a) ² isN, W_(a) ⁴ is NR⁴, and W_(b) ⁵ is CR⁸; (xxix) W_(a) ¹ is CR⁵, W_(a) ² isN, W_(a) ⁴ is NR⁴, and W_(b) ⁵ is CHR⁸; (xxx) W_(a) ¹ is CR⁵, W_(a) ² isN, W_(a) ⁴ is NR⁴, and W_(b) ⁵ is N; (xxxi) W_(a) ¹ is CR⁵, W_(a) ² isN, W_(a) ⁴ is NR⁴, and W_(b) ⁵ is NR⁸; (xxxii) W_(a) ¹ is CR⁵, W_(a) ²is CR⁵, W_(a) ⁴ is S, and W_(b) ⁵ is CHR⁸; (xxxiii) W_(a) ¹ is CR⁵,W_(a) ² is CR⁵, W_(a) ⁴ is S, and W_(b) ⁵ is CR⁸; (xxxiv) W_(a) ¹ isCR⁵, W_(a) ² is CR⁵, W_(a) ⁴ is S, and W_(b) ⁵ is N; and (xxxv) W_(a) isCR⁵, W_(a) ² is CR⁵, W_(a) ⁴ is S, and W_(b) ⁵ is NR⁸.

In some embodiments of the invention, the compound of Formula IX is ofFormula X:

In some embodiments of the invention, the compound of Formula IX is thecompound wherein R⁴ is C₁-C₄alkyl or C₃-C₅cycloalkyl. In someembodiments of the invention, the compound of Formula IX is the compoundwherein R⁴ is methyl or ethyl.

In some embodiments of the invention, the compound of Formula IX is thecompound of Formula XI:

In some embodiments of the invention, the compound of Formula IX is thecompound of Formula XII:

In some embodiments of the invention, the compound of Formula VIII isthe compound of Formula XII or XIII

In some embodiments of the invention, the compound of Formula I, IV, V,VI, VI-A, VI-C, VI-D, 6-A, 6-C1, 6-C2, 6-D, VII, VIII, IX, X, XI, XII,XIII, or XIV is the compound wherein B is a member of the groupconsisting of a moiety of Formula II, wherein W_(c) is aryl includingbut not limited to substituted phenyl, heteroaryl including but notlimited to monocyclic heteroaryl, heterocycloalkyl, or cycloalkyl,heterocycloalkyl, alkyl, including but not limited to a moiety havingthe formula —(CH₂)₂—NR^(a)R^(a), wherein each R^(a) is independentlyhydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl,aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl orheteroarylalkyl, or —NR^(a)R^(a) are combined together to form a cyclicmoiety.

In some embodiments of the invention, the compound of Formula I, IV, V,VI, VI-A, VI-C, VI-D, 6-A, 6-C1, 6-C2, 6-D, VII, VIII, IX, X, XI, XII,XIII, or XIV is the compound wherein R¹ is H, —F, —Cl, —CN, —CH₃,isopropyl, —CF₃, —OCH₃, nitro, or phosphate.

In some embodiments of the invention, the compound of Formula I, IV, V,VI, VI-A, VI-C, VI-D, 6-A, 6-C1, 6-C2, 6-D, VII, VIII, IX, X, XI, XII,XIII, or XIV is the compound wherein R² is a alkyl, halo, hydroxy,cyano, nitro, or phosphate and q is 1 or 2.

In some embodiments of the invention, the compound of Formula I, IV, V,VI, VI-A, VI-C, VI-D, 6-A, 6-C1, 6-C2, 6-D, VII, or VIII is the compoundwherein R³ is —H, halo including but not limited to —Cl or —F, alkylincluding but not limited to —CH₃ or —CH₂CH₃, alkoxy, cycloalkyl, or—CF₃.

In some embodiments of the invention, the compound of Formula IX, or X,R⁴ is selected from —H, methyl, ethyl, n-propyl, iso-propyl,cyclopropyl, cyclobutyl, and cyclopentyl.

In some embodiments of the invention, the compound of Formula I, IV, V,VI, VI-A, VI-C, VI-D, VII, VIII, IX, XI, XII, XIII or XIV is thecompound wherein R⁵ is H, —CN, —NH₂, C₁-C₄alkyl, C₁-C₄alkoxy, —CF₃, NO₂,—CH₃, —CH₂CH₃, —OCH₃, —OCH₂CH₃, or halo, which includes but is notlimited to —Cl or —F.

In some embodiments of the invention, the compound of Formula I, IV, V,VI, VI-A, VI-C, VI-D, VII, or VIII is the compound wherein R⁶ is H, —CN,—NH₂, C₁-C₄alkyl, C₁-C₄alkoxy, —CF₃, NO₂, —CH₃, or halo.

In some embodiments of the invention, the compound of Formula I, IV, V,VI, VI-A, VI-C, VI-D, VII, VIII, IX, or X is the compound wherein R⁷ isH, —CN, —NH₂, C₁-C₄alkyl, C₁-C₄alkoxy, —CF₃, NO₂, —CH₃, —CH₂CH₃, —OCH₃,—OCH₂CH₃, or halo.

In some embodiments of the invention, the compound of Formula I, IV, V,VI, VI-A, VI-C, VI-D, VII, IX, or XII is the compound wherein R⁸ is H,—CN, —NH₂, C₁-C₄alkyl, C₁-C₄alkoxy, —CF₃, NO₂, —CH₃, —CH₂CH₃, —OCH₃,—OCH₂CH₃, or halo.

In some of the embodiments of Formula I, IV, VI, or VII, R⁵, R⁶, R⁷, andR⁸ are hydrogen.

In some embodiments of the invention, the compound of Formula I, IV, V,VI, VI-A, VI-C, VI-D, VII, VIII, IX, X, XI, XII, XIII or XIV is thecompound wherein X is —CH₂—, —CH(CH₂CH₃)— or —CH(CH₃)—, including butnot limited wherein —CH(CH₂CH₃)— or —CH(CH₃)— is in a (S) or (R)stereochemical configuration. In some embodiments of the invention, thecompound of Formula I, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII or XIVis the compound wherein Y is absent, —O—, —NH(R⁹)—, or —S(═O)₂—. In someembodiments of the invention, the compound of Formula I, IV, V, VI, VII,VIII, IX, X, XI, XII, XIII or XIV is the compound wherein R⁹ is methylor hydrogen. In some embodiments of the invention, the compound ofFormula I, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII or XIV is thecompound wherein X—Y is —CH₂N(CH₃), —CH₂—N(CH₂CH₃), —CH(CH₃)NH—,—CH(CH₂CH₃)—NH—, —N(H)CH₂—, —N(CH₂CH₃) CH₂— or —N(CH₃)CH₂—, includingbut not limited to —CH(CH₃)NH— or —CH(CH₂CH₃)—NH having a (S) or (R)stereochemical configuration.

In some embodiments of the invention, the compound of Formula I, IV, V,VI, VII, VIII, IX, X, XI, XII, XIII or XIV is the compound wherein W_(d)is pyrazolopyrimidine including but not limited to4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl or7-amino-2-methyl-2H-pyrazolo[4,3-d]pyrimidin-3-yl, purine including butnot limited to 6-amino-9H-purin-9-yl or 6-methylenyl-9H-purin-6-yl. Insome embodiments of the invention, the compound of Formula I, IV, V, VI,VII, VIII, IX, X, XI, XII, XIII or XIV is the compound wherein thepyrazolopyrimidine is of Formula III:

-   -   wherein R¹¹ is H, alkyl, halo, amino, amido, hydroxy, or alkoxy,        and R¹² is is H, alkyl, alkynyl, alkenyl, halo, aryl, heteroaryl        including but not limited to monocyclic or bicyclic heteroaryl,        heterocycloalkyl, cycloalkyl, cyano, amino, carboxylic acid,        alkoxycarbonyl, or amido.

In another aspect of the invention, a method of inhibiting aphosphatidyl inositol-3 kinase (PI3 kinase), is provided comprising:contacting the PI3 kinase with an effective amount of one or morecompounds disclosed herein. For instance, the step of contactinginvolves the use of one or more compounds of Formula I, IV, V, VI, VII,VIII, IX, X, XI, XII, XIII, and/or XIV. In some embodiments, the step ofcontacting comprises contacting a cell that contains said PI3 kinase. Insome embodiments of the method, the inhibition takes place in a subjectsuffering from a disorder involving malfunctioning of one or more typesof PI3 kinase. Some exemplary diseases involving malfunctioning of PI3kinases are selected from the group consisting of autoimmune diseases,rheumatoid arthritis, respiratory disease, and various types of cancers.Where desired, the compound used in the method has the structure ofFormula 6-A, wherein R¹¹ is amino and R¹² is substituted phenyl.

In some embodiments of the method, the inhibition takes place in asubject suffering from rheumatoid arthritis or a respiratory disease,and wherein the compound has the structure of Formula 6-A, and whereinR¹¹ is amino and R¹² is bicyclic heteroaryl.

In some embodiments, the method comprises administering a secondtherapeutic agent to the subject.

In yet another aspect, the present invention provides a method oftreating a disease manifesting an undesired immune response. The methodcomprises the step of administering to a subject in need thereof, one ormore compounds disclosed herein including compounds of Formula I, IV, V,VI, VII, VIII, IX, X, XI, XII, XIII, and/or XIV, in an amount that iseffective in ameliorating said undesired immune response. In someembodiments, the one or more compounds inhibit T-cell independent B-cellactivation as evidenced by a reduction in production of anti-TNP IgG3 byat least about five folds when administered in an amount less than about30 mg/kg BID dose to a test animal.

In some embodiments, the disease treated is associated with swelling orpain of a joint of a subject. The method can be effective inameliorating one or more rheumatoid arthritis symptoms as evidenced byreduction in mean joint diameter by at least about 10% after 17 daysand/or reduction in ankle diameter by at least 5-10% or more afterseveral days to weeks of treatment, including for example reduction inankle diameter by at least 5% after 7 days of treatment. In anotherembodiment, the undesired immune response is evidenced by enhancedproduction of anti-type II collagen antibodies, and the use of one ormore subject compounds reduces the serum anti-type II collagen level atan ED50 of less than about 10 mg/kg.

In another aspect of the invention, a composition is provided whichcomprises a pharmaceutically acceptable excipient and one or morecompounds of Formula I, IV, V, VI, VII, VIII, IX, X, XI, XII, XIIIand/or XIV. In some of the embodiments of the invention, the compositionis a liquid, solid, semi-solid, gel, or an aerosol form.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 depicts an exemplary protocol for measuring T-cell independentproduction of TNP specific antibodies in vivo.

FIG. 2 depicts the fold reduction in TNP specific IgG3 response toantigens provided by compounds 7 and 53 of formula IV as compared to avehicle control, when administered orally.

FIG. 3 depicts the dose-dependent effect of twice daily oraladministration of compound 53 of formula IV in reducing the increase inankle diameter over time in a collagen-induced developing arthritismodel in rats. Also depicted are the results from non-arthritic controlrats, arthritic control rats administered with a negative controlvehicle, and arthritic control rats treated twice daily withmethotrexate.

FIG. 4 depicts the dose-dependent effect of compounds 7 and 53 offormula IV in improving ankle histopathology when administered in acollagen-induced developing arthritis model in rats. Also depicted arethe results from arthritic control rats administered with negativecontrol vehicle or methotrexate.

FIG. 5 depicts the dose-dependent effect of compounds 7 and 53 offormula IV in improving knee histopathology when administered in acollagen-induced developing arthritis model in rats. Also depicted arethe results from arthritic control rats administered with negativecontrol vehicle or positive control methotrexate.

FIG. 6 depicts the dose-dependent effect of compounds 7 and 53 offormula IV in reducing the level of anti-type II collagen antibodies invivo when administered to a collagen-induced developing arthritis ratmodel. Also depicted are the results from arthritic rats administeredwith negative control vehicle or methotrexate.

FIG. 7 depicts the dose-dependent effect of compound 7 of formula IV onimproving ankle histopathology when administered in collagen-induceddeveloping arthritis model in rats. Also depicted are the results fromarthritic vehicle control rats and methotrexate-treated arthritic rats.

FIG. 8 depicts the dose-dependent effect of compound 53 of formula IVadministered daily on ankle histopathology in a collagen-inducedestablished arthritis model in rats. Also depicted are the results fromarthritic arthritic vehicle control rats and Enbrel-treated arthriticrats.

FIG. 9 depicts the dose-dependent effect of compound 53 of formula IVadministered twice daily on ankle histopathology in a collagen-inducedestablished arthritis model in rats. Also depicted are the results fromarthritic vehicle control rats and Enbrel-treated arthritic rats.

FIG. 10 depicts the dose-dependent effect of compound 53 of formula IVon the increase in average paw volume in an adjuvant induced arthritismodel.

FIG. 11 depicts the effect of compound 53 of formula IV on the averageweight over time of rats in an adjuvant induced arthritis model in rats.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe appended claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference.

As used in the specification and claims, the singular form “a”, “an” and“the” includes plural references unless the context clearly dictatesotherwise.

As used herein, “agent” or “biologically active agent” refers to abiological, pharmaceutical, or chemical compound or other moiety.Non-limiting examples include simple or complex organic or inorganicmolecule, a peptide, a protein, an oligonucleotide, an antibody, anantibody derivative, antibody fragment, a vitamin derivative, acarbohydrate, a toxin, or a chemotherapeutic compound. Various compoundscan be synthesized, for example, small molecules and oligomers (e.g.,oligopeptides and oligonucleotides), and synthetic organic compoundsbased on various core structures. In addition, various natural sourcescan provide compounds for screening, such as plant or animal extracts,and the like. A skilled artisan can readily recognize that there is nolimit as to the structural nature of the agents of the presentinvention.

The term “agonist” as used herein refers to a compound having theability to initiate or enhance a biological function of a targetprotein, whether by inhibiting the activity or expression of the targetprotein. Accordingly, the term “agonist” is defined in the context ofthe biological role of the target polypeptide. While preferred agonistsherein specifically interact with (e.g. bind to) the target, compoundsthat initiate or enhance a biological activity of the target polypeptideby interacting with other members of the signal transduction pathway ofwhich the target polypeptide is a member are also specifically includedwithin this definition.

The terms “antagonist” and “inhibitor” are used interchangeably, andthey refer to a compound having the ability to inhibit a biologicalfunction of a target protein, whether by inhibiting the activity orexpression of the target protein. Accordingly, the terms “antagonist”and “inhibitors” are defined in the context of the biological role ofthe target protein. While preferred antagonists herein specificallyinteract with (e.g. bind to) the target, compounds that inhibit abiological activity of the target protein by interacting with othermembers of the signal transduction pathway of which the target proteinis a member are also specifically included within this definition. Apreferred biological activity inhibited by an antagonist is associatedwith the development, growth, or spread of a tumor, or an undesiredimmune response as manifested in autoimmune disease.

An “anti-cancer agent”, “anti-tumor agent” or “chemotherapeutic agent”refers to any agent useful in the treatment of a neoplastic condition.One class of anti-cancer agents comprises chemotherapeutic agents.“Chemotherapy” means the administration of one or more chemotherapeuticdrugs and/or other agents to a cancer patient by various methods,including intravenous, oral, intramuscular, intraperitoneal,intravesical, subcutaneous, transdermal, buccal, or inhalation or in theform of a suppository.

The term “cell proliferation” refers to a phenomenon by which the cellnumber has changed as a result of division. This term also encompassescell growth by which the cell morphology has changed (e.g., increased insize) consistent with a proliferative signal.

The term “co-administration,” “administered in combination with,” andtheir grammatical equivalents, as used herein, encompassesadministration of two or more agents to an animal so that both agentsand/or their metabolites are present in the animal at the same time.Co-administration includes simultaneous administration in separatecompositions, administration at different times in separatecompositions, or administration in a composition in which both agentsare present.

The term “effective amount” or “therapeutically effective amount” refersto that amount of a compound described herein that is sufficient toeffect the intended application including but not limited to diseasetreatment, as defined below. The therapeutically effective amount mayvary depending upon the intended application (in vitro or in vivo), orthe subject and disease condition being treated, e.g., the weight andage of the subject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofordinary skill in the art. The term also applies to a dose that willinduce a particular response in target cells, e.g. reduction of plateletadhesion and/or cell migration. The specific dose will vary depending onthe particular compounds chosen, the dosing regimen to be followed,whether it is administered in combination with other compounds, timingof administration, the tissue to which it is administered, and thephysical delivery system in which it is carried.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably herein. These terms refers to anapproach for obtaining beneficial or desired results including but notlimited to therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the patient, notwithstanding that thepatient may still be afflicted with the underlying disorder. Forprophylactic benefit, the compositions may be administered to a patientat risk of developing a particular disease, or to a patient reportingone or more of the physiological symptoms of a disease, even though adiagnosis of this disease may not have been made.

A “therapeutic effect,” as that term is used herein, encompasses atherapeutic benefit and/or a prophylactic benefit as described above. Aprophylactic effect includes delaying or eliminating the appearance of adisease or condition, delaying or eliminating the onset of symptoms of adisease or condition, slowing, halting, or reversing the progression ofa disease or condition, or any combination thereof.

The term “pharmaceutically acceptable salt” refers to salts derived froma variety of organic and inorganic counter ions well known in the art.Pharmaceutically acceptable acid addition salts can be formed withinorganic acids and organic acids. Inorganic acids from which salts canbe derived include, for example, hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acidsfrom which salts can be derived include, for example, acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and thelike. Pharmaceutically acceptable base addition salts can be formed withinorganic and organic bases. Inorganic bases from which salts can bederived include, for example, sodium, potassium, lithium, ammonium,calcium, magnesium, iron, zinc, copper, manganese, aluminum, and thelike. Organic bases from which salts can be derived include, forexample, primary, secondary, and tertiary amines, substituted aminesincluding naturally occurring substituted amines, cyclic amines, basicion exchange resins, and the like, specifically such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine, andethanolamine. In some embodiments, the pharmaceutically acceptable baseaddition salt is chosen from ammonium, potassium, sodium, calcium, andmagnesium salts.

“Pharmaceutically acceptable carrier” or “pharmaceutically acceptableexcipient” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions of theinvention is contemplated. Supplementary active ingredients can also beincorporated into the compositions.

“Signal transduction” is a process during which stimulatory orinhibitory signals are transmitted into and within a cell to elicit anintracellular response. A modulator of a signal transduction pathwayrefers to a compound which modulates the activity of one or morecellular proteins mapped to the same specific signal transductionpathway. A modulator may augment (agonist) or suppress (antagonist) theactivity of a signaling molecule.

The term “selective inhibition” or “selectively inhibit” as applied to abiologically active agent refers to the agent's ability to selectivelyreduce the target signaling activity as compared to off-target signalingactivity, via direct or interact interaction with the target.

The term “B-ALL” as used herein refers to B-cell Acute LymphoblasticLeukemia.

“Subject” refers to an animal, such as a mammal, for example a human.The methods described herein can be useful in both human therapeuticsand veterinary applications. In some embodiments, the patient is amammal, and in some embodiments, the patient is human.

“Radiation therapy” means exposing a patient, using routine methods andcompositions known to the practitioner, to radiation emitters such asalpha-particle emitting radionuclides (e.g., actinium and thoriumradionuclides), low linear energy transfer (LET) radiation emitters(i.e. beta emitters), conversion electron emitters (e.g. strontium-89and samarium-153-EDTMP, or high-energy radiation, including withoutlimitation x-rays, gamma rays, and neutrons.

“Prodrug” is meant to indicate a compound that may be converted underphysiological conditions or by solvolysis to a biologically activecompound described herein. Thus, the term “prodrug” refers to aprecursor of a biologically active compound that is pharmaceuticallyacceptable. A prodrug may be inactive when administered to a subject,but is converted in vivo to an active compound, for example, byhydrolysis. The prodrug compound often offers advantages of solubility,tissue compatibility or delayed release in a mammalian organism (see,e.g., Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier,Amsterdam). A discussion of prodrugs is provided in Higuchi, T., et al.,“Pro-drugs as Novel Delivery Systems,” A.C.S. Symposium Series, Vol. 14,and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche,American Pharmaceutical Association and Pergamon Press, 1987, both ofwhich are incorporated in full by reference herein. The term “prodrug”is also meant to include any covalently bonded carriers, which releasethe active compound in vivo when such prodrug is administered to amammalian subject. Prodrugs of an active compound, as described herein,may be prepared by modifying functional groups present in the activecompound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent active compound. Prodrugsinclude compounds wherein a hydroxy, amino or mercapto group is bondedto any group that, when the prodrug of the active compound isadministered to a mammalian subject, cleaves to form a free hydroxy,free amino or free mercapto group, respectively. Examples of prodrugsinclude, but are not limited to, acetate, formate and benzoatederivatives of an alcohol or acetamide, formamide and benzamidederivatives of an amine functional group in the active compound and thelike.

The term “in vivo” refers to an event that takes place in a subject'sbody.

The term “in vitro” refers to an event that takes places outside of asubject's body. For example, an in vitro assay encompasses any assay runoutside of a subject assay. In vitro assays encompass cell-based assaysin which cells alive or dead are employed. In vitro assays alsoencompass a cell-free assay in which no intact cells are employed.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of atoms that constitutesuch compounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe present invention, whether radioactive or not, are encompassedwithin the scope of the present invention.

When ranges are used herein for physical properties, such as molecularweight, or chemical properties, such as chemical formulae, allcombinations and subcombinations of ranges and specific embodimentstherein are intended to be included. The term “about” when referring toa number or a numerical range means that the number or numerical rangereferred to is an approximation within experimental variability (orwithin statistical experimental error), and thus the number or numericalrange may vary from, for example, between 1% and 15% of the statednumber or numerical range. The term “comprising” (and related terms suchas “comprise” or “comprises” or “having” or “including”) includes thoseembodiments, for example, an embodiment of any composition of matter,composition, method, or process, or the like, that “consist of” or“consist essentially of” the described features.

The following abbreviations and terms have the indicated meaningsthroughout PI3-K=Phosphoinositide 3-kinase; PI=phosphatidylinositol;PDK=Phosphoinositide Dependent Kinase; DNA-PK=Deoxyribose Nucleic AcidDependent Protein Kinase; PTEN=Phosphatase and Tensin homolog deleted onchromosome Ten; PIKK=Phosphoinositide Kinase Like Kinase; AIDS=AcquiredImmuno Deficiency Syndrome; HIV=Human Immunodeficiency Virus; MeI=MethylIodide; POCl₃=Phosphorous Oxychloride; KCNS=Potassium IsoThiocyanate;TLC=Thin Layer Chromatography; MeOH=Methanol; and CHCl₃=Chloroform.

Abbreviations used herein have their conventional meaning within thechemical and biological arts.

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, containing nounsaturation, having from one to ten carbon atoms (e.g., C₁-C₁₀ alkyl).Whenever it appears herein, a numerical range such as “1 to 10” refersto each integer in the given range; e.g., “1 to 10 carbon atoms” meansthat the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3carbon atoms, etc., up to and including 10 carbon atoms, although thepresent definition also covers the occurrence of the term “alkyl” whereno numerical range is designated. In some embodiments, it is a C₁-C₄alkyl group. Typical alkyl groups include, but are in no way limited to,methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butylisobutyl, tertiary butyl, pentyl, isopentyl, neopentyl, hexyl, septyl,octyl, nonyl, decyl, and the like. The alkyl is attached to the rest ofthe molecule by a single bond, for example, methyl (Me), ethyl (Et),n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl,1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, and the like.Unless stated otherwise specifically in the specification, an alkylgroup is optionally substituted by one or more of substituents whichindependently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy,halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl,—OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂ whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Alkylaryl” refers to an -(alkyl)aryl radical where aryl and alkyl areas disclosed herein and which are optionally substituted by one or moreof the substituents described as suitable substituents for aryl andalkyl respectively.

“Alkylhetaryl” refers to an -(alkyl)hetaryl radical where hetaryl andalkyl are as disclosed herein and which are optionally substituted byone or more of the substituents described as suitable substituents foraryl and alkyl respectively.

“Alkylheterocycloalkyl” refers to an -(alkyl) heterocycyl radical wherealkyl and heterocycloalkyl are as disclosed herein and which areoptionally substituted by one or more of the substituents described assuitable substituents for heterocycloalkyl and alkyl respectively.

An “alkene” moiety refers to a group consisting of at least two carbonatoms and at least one carbon-carbon double bond, and an “alkyne” moietyrefers to a group consisting of at least two carbon atoms and at leastone carbon-carbon triple bond. The alkyl moiety, whether saturated orunsaturated, may be branched, straight chain, or cyclic.

“Alkenyl” refers to a straight or branched hydrocarbon chain radicalgroup consisting solely of carbon and hydrogen atoms, containing atleast one double bond, and having from two to ten carbon atoms (i.e.C₂-C₁₀ alkenyl). Whenever it appears herein, a numerical range such as“2 to 10” refers to each integer in the given range; e.g., “2 to 10carbon atoms” means that the alkenyl group may consist of 2 carbonatoms, 3 carbon atoms, etc., up to and including 10 carbon atoms. Incertain embodiments, an alkenyl comprises two to eight carbon atoms. Inother embodiments, an alkenyl comprises two to five carbon atoms (e.g.,C₂-C₅ alkenyl). The alkenyl is attached to the rest of the molecule by asingle bond, for example, ethenyl (i.e., vinyl), prop-1-enyl (i.e.,allyl), but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and the like. Unlessstated otherwise specifically in the specification, an alkenyl group isoptionally substituted by one or more substituents which independentlyare: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano,trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a),—SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Alkenyl-cycloalkyl” refers to refers to an -(alkenyl)cycloalkyl radicalwhere alkenyl and cyclo alkyl are as disclosed herein and which areoptionally substituted by one or more of the substituents described assuitable substituents for alkenyl and cycloalkyl respectively.

“Alkynyl” refers to a straight or branched hydrocarbon chain radicalgroup consisting solely of carbon and hydrogen atoms, containing atleast one triple bond, having from two to ten carbon atoms (i.e. C₂-C₁₀alkynyl). Whenever it appears herein, a numerical range such as “2 to10” refers to each integer in the given range; e.g., “2 to 10 carbonatoms” means that the alkynyl group may consist of 2 carbon atoms, 3carbon atoms, etc., up to and including 10 carbon atoms. In certainembodiments, an alkynyl comprises two to eight carbon atoms. In otherembodiments, an alkynyl has two to five carbon atoms (e.g., C₂-C₅alkynyl). The alkynyl is attached to the rest of the molecule by asingle bond, for example, ethynyl, propynyl, butynyl, pentynyl, hexynyl,and the like. Unless stated otherwise specifically in the specification,an alkynyl group is optionally substituted by one or more substituentswhich independently are: alkyl, heteroalkyl, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a), —SR^(a),—OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂,—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Alkynyl-cycloalkyl” refers to refers to an -(alkynyl)cycloalkyl radicalwhere alkynyl and cycloalkyl are as disclosed herein and which areoptionally substituted by one or more of the substituents described assuitable substituents for alkynyl and cycloalkyl respectively.

“Carboxaldehyde” refers to a —(C═O)H radical.

“Carboxyl” refers to a —(C═O)OH radical.

“Cyano” refers to a —CN radical.

“Cycloalkyl” refers to a monocyclic or polycyclic radical that containsonly carbon and hydrogen, and may be saturated, or partiallyunsaturated. Cycloalkyl groups include groups having from 3 to 10 ringatoms (i.e. C₂-C₁₀ cycloalkyl). Whenever it appears herein, a numericalrange such as “3 to 10” refers to each integer in the given range; e.g.,“3 to 10 carbon atoms” means that the cycloalkyl group may consist of 3carbon atoms, etc., up to and including 10 carbon atoms. In someembodiments, it is a C₃-C₈ cycloalkyl radical. In some embodiments, itis a C₃-C₅ cycloalkyl radical. Illustrative examples of cycloalkylgroups include, but are not limited to the following moieties:cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl,cyclohexenyl, cycloseptyl, cyclooctyl, cyclononyl, cyclodecyl,norbornyl, and the like. Unless stated otherwise specifically in thespecification, a cycloalkyl group is optionally substituted by one ormore substituents which independently are: alkyl, heteroalkyl, alkenyl,alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a), —SR^(a),—OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂,—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Cycloalkyl-alkenyl” refers to a -(cycloalkyl) alkenyl radical wherecycloalkyl and heterocycloalkyl are as disclosed herein and which areoptionally substituted by one or more of the substituents described assuitable substituents for heterocycloalkyl and cycloalkyl respectively.

“Cycloalkyl-heterocycloalkyl” refers to a -(cycloalkyl) heterocycylradical where cycloalkyl and heterocycloalkyl are as disclosed hereinand which are optionally substituted by one or more of the substituentsdescribed as suitable substituents for heterocycloalkyl and cycloalkylrespectively.

“Cycloalkyl-heteroaryl” refers to a -(cycloalkyl) heteroaryl radicalwhere cycloalkyl and heterocycloalkyl are as disclosed herein and whichare optionally substituted by one or more of the substituents describedas suitable substituents for heterocycloalkyl and cycloalkylrespectively.

The term “alkoxy” refers to the group —O-alkyl, including from 1 to 8carbon atoms of a straight, branched, cyclic configuration andcombinations thereof attached to the parent structure through an oxygen.Examples include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy,cyclohexyloxy and the like. “Lower alkoxy” refers to alkoxy groupscontaining one to six carbons. In some embodiments, C₁-C₄ alkyl, is analkyl group which encompasses both straight and branched chain alkyls offrom 1 to 4 carbon atoms.

The term “substituted alkoxy” refers to alkoxy wherein the alkylconstituent is substituted (i.e., —O-(substituted alkyl)). Unless statedotherwise specifically in the specification, the alkyl moiety of analkoxy group is optionally substituted by one or more substituents whichindependently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy,halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl,—OR^(a), SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

The term “alkoxycarbonyl” refers to a group of the formula(alkoxy)(C═O)— attached through the carbonyl carbon wherein the alkoxygroup has the indicated number of carbon atoms. Thus a C₁-C₆alkoxycarbonyl group is an alkoxy group having from 1 to 6 carbon atomsattached through its oxygen to a carbonyl linker. “Lower alkoxycarbonyl”refers to an alkoxycarbonyl group wherein the alkoxy group is a loweralkoxy group. In some embodiments, C₁-C₄ alkoxy, is an alkoxy groupwhich encompasses both straight and branched chain alkoxy groups of from1 to 4 carbon atoms.

The term “substituted alkoxycarbonyl” refers to the group (substitutedalkyl)-O—C(O)— wherein the group is attached to the parent structurethrough the carbonyl functionality. Unless stated otherwise specificallyin the specification, the alkyl moiety of an alkoxycarbonyl group isoptionally substituted by one or more substituents which independentlyare: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano,trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a),SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, ayl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl,heteroaryl or heteroarylalkyl.

“Acyl” refers to the groups (alkyl)-C(O)—, (aryl)-C(O)—,(heteroaryl)-C(O)—, (heteroalkyl)-C(O)—, and (heterocycloalkyl)-C(O)—,wherein the group is attached to the parent structure through thecarbonyl functionality. In some embodiments, it is a C₁-C₁₀ acyl radicalwhich refers to the total number of chain or ring atoms of the alkyl,aryl, heteroaryl or heterocycloalkyl portion of the acyloxy group plusthe carbonyl carbon of acyl, i.e three other ring or chain atoms pluscarbonyl. If the R radical is heteroaryl or heterocycloalkyl, the heteroring or chain atoms contribute to the total number of chain or ringatoms. Unless stated otherwise specifically in the specification, the“R” of an acyloxy group is optionally substituted by one or moresubstituents which independently are: alkyl, heteroalkyl, alkenyl,alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a), SR^(a),—OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂,—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, ayl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl,heteroaryl or heteroarylalkyl.

“Acyloxy” refers to a R(C═O)O— radical wherein “R” is alkyl, aryl,heteroaryl, heteroalkyl, or heterocycloalkyl, which are as describedherein. In some embodiments, it is a C₁-C₄ acyloxy radical which refersto the total number of chain or ring atoms of the alkyl, aryl,heteroaryl or heterocycloalkyl portion of the acyloxy group plus thecarbonyl carbon of acyl, i.e three other ring or chain atoms pluscarbonyl. If the R radical is heteroaryl or heterocycloalkyl, the heteroring or chain atoms contribute to the total number of chain or ringatoms. Unless stated otherwise specifically in the specification, the“R” of an acyloxy group is optionally substituted by one or moresubstituents which independently are: alkyl, heteroalkyl, alkenyl,alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a), —SR^(a),—OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂,—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2-S(O)_(t)OR^(a) (where t is 1or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Amino” or “amine” refers to a —N(R^(a))₂ radical group, where eachR^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, unless statedotherwise specifically in the specification. When a —N(R^(a))₂ group hastwo R^(a) other than hydrogen they can be combined with the nitrogenatom to form a 4-, 5-, 6-, or 7-membered ring. For example, —N(R^(a))₂is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. Unless stated otherwise specifically in thespecification, an amino group is optionally substituted by one or moresubstituents which independently are: alkyl, heteroalkyl, alkenyl,alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a), —SR^(a),—OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂,—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl and each of thesemoieties may be optionally substituted as defined herein.

The term “substituted amino” also refers to N-oxides of the groups—NHR^(d), and NR^(d)R^(d) each as described above. N-oxides can beprepared by treatment of the corresponding amino group with, forexample, hydrogen peroxide or m-chloroperoxybenzoic acid. The personskilled in the art is familiar with reaction conditions for carrying outthe N-oxidation.

“Amide” or “amido” refers to a chemical moiety with formula —C(O)N(R)₂or —NHC(O)R, where R is selected from the group consisting of hydrogen,alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) andheteroalicyclic (bonded through a ring carbon), each of which moiety mayitself be optionally substituted. In some embodiments it is a C₁-C₄amido or amide radical, which includes the amide carbonyl in the totalnumber of carbons in the radical. The R₂ of —N(R)₂ of the amide mayoptionally be taken together with the nitrogen to which it is attachedto form a 4-, 5-, 6-, or 7-membered ring. Unless stated otherwisespecifically in the specification, an amido group is optionallysubstituted independently by one or more of the substituents asdescribed herein for alkyl, cycloalkyl, aryl, heteroaryl, orheterocycloalkyl. An amide may be an amino acid or a peptide moleculeattached to a compound of Formula (I), thereby forming a prodrug. Anyamine, hydroxy, or carboxyl side chain on the compounds described hereincan be amidified. The procedures and specific groups to make such amidesare known to those of skill in the art and can readily be found inreference sources such as Greene and Wuts, Protective Groups in OrganicSynthesis, 3.sup.rd Ed., John Wiley & Sons, New York, N.Y., 1999, whichis incorporated herein by reference in its entirety.

“Aromatic” or “aryl” refers to an aromatic radical with six to ten ringatoms (e.g., C₆-C₁₀ aromatic or C₆-C₁₀ aryl) which has at least one ringhaving a conjugated pi electron system which is carbocyclic (e.g.,phenyl, fluorenyl, and naphthyl). Bivalent radicals formed fromsubstituted benzene derivatives and having the free valences at ringatoms are named as substituted phenylene radicals. Bivalent radicalsderived from univalent polycyclic hydrocarbon radicals whose names endin “-yl” by removal of one hydrogen atom from the carbon atom with thefree valence are named by adding “-idene” to the name of thecorresponding univalent radical, e.g., a naphthyl group with two pointsof attachment is termed naphthylidene. Whenever it appears herein, anumerical range such as “6 to 10” refers to each integer in the givenrange; e.g., “6 to 10 ring atoms” means that the aryl group may consistof 6 ring atoms, 7 ring atoms, etc., up to and including 10 ring atoms.The term includes monocyclic or fused-ring polycyclic (i.e., rings whichshare adjacent pairs of ring atoms) groups. Unless stated otherwisespecifically in the specification, an aryl moiety is optionallysubstituted by one or more substituents which are independently: alkyl,heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano,trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a),—SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Aralkyl” or “arylalkyl” refers to an (aryl)alkyl—radical where aryl andalkyl are as disclosed herein and which are optionally substituted byone or more of the substituents described as suitable substituents foraryl and alkyl respectively.

“Ester” refers to a chemical radical of formula —COOR, where R isselected from the group consisting of alkyl, cycloalkyl, aryl,heteroaryl (bonded through a ring carbon) and heteroalicyclic (bondedthrough a ring carbon). Any amine, hydroxy, or carboxyl side chain onthe compounds described herein can be esterified. The procedures andspecific groups to make such esters are known to those of skill in theart and can readily be found in reference sources such as Greene andWuts, Protective Groups in Organic Synthesis, 3.sup.rd Ed., John Wiley &Sons, New York, N.Y., 1999, which is incorporated herein by reference inits entirety. Unless stated otherwise specifically in the specification,an ester group is optionally substituted by one or more substituentswhich independently are: alkyl, heteroalkyl, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a), —SR^(a),—OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂,—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Fluoroalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more fluoro radicals, as defined above, forexample, trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl,1-fluoromethyl-2-fluoroethyl, and the like. The alkyl part of thefluoroalkyl radical may be optionally substituted as defined above foran alkyl group.

“Halo”, “halide”, or, alternatively, “halogen” means fluoro, chloro,bromo or iodo. The terms “haloalkyl,” “haloalkenyl,” “haloalkynyl” and“haloalkoxy” include alkyl, alkenyl, alkynyl and alkoxy structures thatare substituted with one or more halo groups or with combinationsthereof. For example, the terms “fluoroalkyl” and “fluoroalkoxy” includehaloalkyl and haloalkoxy groups, respectively, in which the halo isfluorine.

“Heteroalkyl” “heteroalkenyl” and “heteroalkynyl” include optionallysubstituted alkyl, alkenyl and alkynyl radicals and which have one ormore skeletal chain atoms selected from an atom other than carbon, e.g.,oxygen, nitrogen, sulfur, phosphorus or combinations thereof. Anumerical range may be given, e.g. C₁-C₄ heteroalkyl which refers to thechain length in total, which in this example is 4 atoms long. Forexample, a —CH₂OCH₂CH₃ radical is referred to as a “C₄” heteroalkyl,which includes the heteroatom center in the atom chain lengthdescription. Connection to the rest of the molecule may be througheither a heteroatom or a carbon in the heteroalkyl chain. A heteroalkylgroup may be substituted with one or more substituents whichindependently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy,halo, cyano, nitro, oxo, thioxo, trimethylsilanyl, —OR^(a), —SR^(a),—OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))S(O)_(t)R^(a) (where tis 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2), —S(O)_(t)N(R^(a))₂(where t is 1 or 2), or PO₃(R^(a))₂, where each R^(a) is independentlyhydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl,aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl orheteroarylalkyl.

“Heteroalkylaryl” refers “to an -(heteroalkyl)aryl radical whereheteroalkyl and aryl are as disclosed herein and which are optionallysubstituted by one or more of the substituents described as suitablesubstituents for heteroalkyl and aryl respectively.

“Heteroalkylheteroaryl” refers “to an -(heteroalkyl)heteroaryl radicalwhere heteroalkyl and heteroaryl are as disclosed herein and which areoptionally substituted by one or more of the substituents described assuitable substituents for heteroalkyl and heteroaryl respectively

“Heteroalkylheterocycloalkyl” refers “to an-(heteroalkyl)heterocycloalkyl radical where heteroalkyl and heteroarylare as disclosed herein and which are optionally substituted by one ormore of the substituents described as suitable substituents forheteroalkyl and heterocycloalkyl respectively

“Heteroalkylcycloalkyl” refers “to an -(heteroalkyl) cycloalkyl radicalwhere heteroalkyl and cycloalkyl are as disclosed herein and which areoptionally substituted by one or more of the substituents described assuitable substituents for heteroalkyl and cycloalkyl respectively.

“Heteroaryl” or, alternatively, “heteroaromatic” refers to a 5- to18-membered aromatic radical (e.g., C₅-C₁₃ heteroaryl) that includes oneor more ring heteroatoms selected from nitrogen, oxygen and sulfur, andwhich may be a monocyclic, bicyclic, tricyclic or tetracyclic ringsystem. Whenever it appears herein, a numerical range such as “5 to 18”refers to each integer in the given range; e.g., “5 to 18 ring atoms”means that the heteroaryl group may consist of 5 ring atoms, 6 ringatoms, etc., up to and including 18 ring atoms. Bivalent radicalsderived from univalent heteroaryl radicals whose names end in “-yl” byremoval of one hydrogen atom from the atom with the free valence arenamed by adding “-idene” to the name of the corresponding univalentradical, e.g., a pyridyl group with two points of attachment is apyridylidene. An N-containing “heteroaromatic” or “heteroaryl” moietyrefers to an aromatic group in which at least one of the skeletal atomsof the ring is a nitrogen atom. The polycyclic heteroaryl group may befused or non-fused. The heteroatom(s) in the heteroaryl radical isoptionally oxidized. One or more nitrogen atoms, if present, areoptionally quaternized. The heteroaryl is attached to the rest of themolecule through any atom of the ring(s). Examples of heteroarylsinclude, but are not limited to, azepinyl, acridinyl, benzimidazolyl,benzindolyl, 1,3-benzodioxolyl, benzofuranyl, benzooxazolyl,benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl,benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzoxazolyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzofurazanyl,benzothiazolyl, benzothienyl (benzothiophenyl),benzothieno[3,2-d]pyrimidinyl, benzotriazolyl,benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl,cyclopenta[d]pyrimidinyl,6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl,5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl,6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl,dibenzothiophenyl, furanyl, furazanyl, furanonyl, furo[3,2-c]pyridinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl,indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl,isoquinolyl, indolizinyl, isoxazolyl,5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl,1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl,5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrrolyl,phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl,purinyl, pyranyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl,pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl,pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl,quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl,5,6,7,8-tetrahydroquinazolinyl,5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl,6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl,5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl,thiapyranyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl,thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl, and thiophenyl (i.e.thienyl). Unless stated otherwise specifically in the specification, aheteraryl moiety is optionally substituted by one or more substituentswhich are independently: alkyl, heteroalkyl, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, nitro, oxo, thioxo,trimethylsilanyl, —OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂,—C(O)R^(a), —C(O)OR^(a), —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2),—S(O)_(t)OR^(a) (where t is 1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or2), or PO₃(R^(a))₂, where each R^(a) is independently hydrogen, alkyl,fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl,heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

Substituted heteroaryl also includes ring systems substituted with oneor more oxide (—O—) substituents, such as pyridinyl N-oxides.

“Heteroarylalkyl” refers to a moiety having an aryl moiety, as describedherein, connected to an alkylene moiety, as described herein, whereinthe connection to the remainder of the molecule is through the alkylenegroup.

“Heterocycloalkyl” refers to a stable 3- to 18-membered non-aromaticring radical that comprises two to twelve carbon atoms and from one tosix heteroatoms selected from nitrogen, oxygen and sulfur. Whenever itappears herein, a numerical range such as “3 to 18” refers to eachinteger in the given range; e.g., “3 to 18 ring atoms” means that theheterocycloalkyl group may consist of 3 ring atoms, 4 ring atoms, etc.,up to and including 18 ring atoms. In some embodiments, it is a C₅-C₁₀heterocycloalkyl. In some embodiments, it is a C₄-C₁₀ heterocycloalkyl.In some embodiments, it is a C₃-C₁₀ heterocycloalkyl. Unless statedotherwise specifically in the specification, the heterocycloalkylradical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system,which may include fused or bridged ring systems. The heteroatoms in theheterocycloalkyl radical may be optionally oxidized. One or morenitrogen atoms, if present, are optionally quaternized. Theheterocycloalkyl radical is partially or fully saturated. Theheterocycloalkyl may be attached to the rest of the molecule through anyatom of the ring(s). Examples of such heterocycloalkyl radicals include,but are not limited to, dioxolanyl, thienyl[1,3]dithianyl,decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl,isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl,2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl,piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl,quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl,tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl,1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless statedotherwise specifically in the specification, a heterocycloalkyl moietyis optionally substituted by one or more substituents whichindependently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy,halo, cyano, nitro, oxo, thioxo, trimethylsilanyl, —OR^(a), —SR^(a),—OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))S(O)_(t)R^(a) (where tis 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2), —S(O)_(t)N(R^(a))₂(where t is 1 or 2), or PO₃(R^(a))₂, where each R^(a) is independentlyhydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl,aralkyl, heterocycloalkyl, heteroaryl or heteroarylalkyl.

“Heterocycloalkyl” also includes bicyclic ring systems wherein onenon-aromatic ring, usually with 3 to 7 ring atoms, contains at least 2carbon atoms in addition to 1-3 heteroatoms independently selected fromoxygen, sulfur, and nitrogen, as well as combinations comprising atleast one of the foregoing heteroatoms; and the other ring, usually with3 to 7 ring atoms, optionally contains 1-3 heteroatoms independentlyselected from oxygen, sulfur, and nitrogen and is not aromatic.

“Isomers” are different compounds that have the same molecular formula.“Stereoisomers” are isomers that differ only in the way the atoms arearranged in space. “Enantiomers” are a pair of stereoisomers that arenon-superimposable mirror images of each other. A 1:1 mixture of a pairof enantiomers is a “racemic” mixture. The term “(.±.)” is used todesignate a racemic mixture where appropriate. “Diastereoisomers” arestereoisomers that have at least two asymmetric atoms, but which are notmirror-images of each other. The absolute stereochemistry is specifiedaccording to the Cahn-Ingold-Prelog R-S system. When a compound is apure enantiomer the stereochemistry at each chiral carbon can bespecified by either R or S. Resolved compounds whose absoluteconfiguration is unknown can be designated (+) or (−) depending on thedirection (dextro- or levorotatory) which they rotate plane polarizedlight at the wavelength of the sodium D line. Certain of the compoundsdescribed herein contain one or more asymmetric centers and can thusgive rise to enantiomers, diastereomers, and other stereoisomeric formsthat can be defined, in terms of absolute stereochemistry, as (R)- or(S)-. The present chemical entities, pharmaceutical compositions andmethods are meant to include all such possible isomers, includingracemic mixtures, optically pure forms and intermediate mixtures.Optically active (R)- and (S)-isomers can be prepared using chiralsynthons or chiral reagents, or resolved using conventional techniques.When the compounds described herein contain olefinic double bonds orother centers of geometric asymmetry, and unless specified otherwise, itis intended that the compounds include both E and Z geometric isomers.

“Moiety” refers to a specific segment or functional group of a molecule.Chemical moieties are often recognized chemical entities embedded in orappended to a molecule.

“Nitro” refers to the —NO₂ radical.

“Oxa” refers to the —O— radical.

“Oxo” refers to the ═O radical.

“Tautomers” are structurally distinct isomers that interconvert bytautomerization. “Tautomerization” is a form of isomerization andincludes prototropic or proton-shift tautomerization, which isconsidered a subset of acid-base chemistry. “Prototropictautomerization” or “proton-shift tautomerization” involves themigration of a proton accompanied by changes in bond order, often theinterchange of a single bond with an adjacent double bond. Wheretautomerization is possible (e.g. in solution), a chemical equilibriumof tautomers can be reached. An example of tautomerization is keto-enoltautomerization. A specific example of keto-enol tautomerization is theinterconversion of pentane-2,4-dione and 4-hydroxypent-3-en-2-onetautomers. Another example of tautomerization is phenol-ketotautomerization. A specific example of phenol-keto tautomerization isthe interconversion of pyridin-4-ol and pyridin-4(1H)-one tautomers.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of atoms that constitutesuch compounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe present invention, whether radioactive or not, are encompassedwithin the scope of the present invention.

A “leaving group or atom” is any group or atom that will, under thereaction conditions, cleave from the starting material, thus promotingreaction at a specified site. Suitable examples of such groups unlessotherwise specified are halogen atoms, mesyloxy,p-nitrobenzensulphonyloxy and tosyloxy groups.

“Protecting group” has the meaning conventionally associated with it inorganic synthesis, i.e. a group that selectively blocks one or morereactive sites in a multifunctional compound such that a chemicalreaction can be carried out selectively on another unprotected reactivesite and such that the group can readily be removed after the selectivereaction is complete. A variety of protecting groups are disclosed, forexample, in T. H. Greene and P. G. M. Wuts, Protective Groups in OrganicSynthesis, Third Edition, John Wiley & Sons, New York (1999). Forexample, a hydroxy protected form is where at least one of the hydroxygroups present in a compound is protected with a hydroxy protectinggroup. Likewise, amines and other reactive groups may similarly beprotected.

“Solvate” refers to a compound (e.g., a compound selected from Formula Ior a pharmaceutically acceptable salt thereof) in physical associationwith one or more molecules of a pharmaceutically acceptable solvent. Itwill be understood that “a compound of Formula I” encompass the compoundof Formula I and solvates of the compound, as well as mixtures thereof.

“Substituted” means that the referenced group may be substituted withone or more additional group(s) individually and independently selectedfrom acyl, alkyl, alkylaryl, cycloalkyl, aralkyl, aryl, carbohydrate,carbonate, heteroaryl, heterocycloalkyl, hydroxy, alkoxy, aryloxy,mercapto, alkylthio, arylthio, cyano, halo, carbonyl, ester,thiocarbonyl, isocyanato, thiocyanato, isothiocyanato, nitro, oxo,perhaloalkyl, perfluoroalkyl, phosphate, silyl, sulfinyl, sulfonyl,sulfonamidyl, sulfoxyl, sulfonate, urea, and amino, including mono- anddi-substituted amino groups, and the protected derivatives thereof.Di-substituted amino groups encompass those which form a ring togetherwith the nitrogen of the amino group, such as for instance, morpholino.The substituents themselves may be substituted, for example, acycloalkyl substituent may have a halide substituted at one or more ringcarbons, and the like. The protecting groups that may form theprotective derivatives of the above substituents are known to those ofskill in the art and may be found in references such as Greene and Wuts,above.

“Sulfanyl” refers to the groups: —S-(optionally substituted alkyl),—S-(optionally substituted aryl), —S-(optionally substitutedheteroaryl), and —S-(optionally substituted heterocycloalkyl).

“Sulfinyl” refers to the groups: —S(O)—H, —S(O)-(optionally substitutedalkyl), —S(O)-(optionally substituted amino), —S(O)-(optionallysubstituted aryl), —S(O)-(optionally substituted heteroaryl), and—S(O)-(optionally substituted heterocycloalkyl).

“Sulfonyl” refers to the groups: —S(O₂)—H, —S(O₂)-(optionallysubstituted alkyl), —S(O₂)-(optionally substituted amino),—S(O₂)-(optionally substituted aryl), —S(O₂)-(optionally substitutedheteroaryl), and —S(O₂)-(optionally substituted heterocycloalkyl).

“Sulfonamidyl” or “sulfonamido” refers to a —S(═O)₂—NRR radical, whereeach R is selected independently from the group consisting of hydrogen,alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) andheteroalicyclic (bonded through a ring carbon). The R groups in —NRR ofthe —S(═O)₂—NRR radical may be taken together with the nitrogen to whichit is attached to form a 4-, 5-, 6-, or 7-membered ring. In someembodiments, it is a C₁-C₁₀ sulfonamido, wherein each R in sulfonamidocontains 1 carbon, 2 carbons, 3 carbons, or 4 carbons total. Asulfonamido group is optionally substituted by one or more of thesubstituents described for alkyl, cycloalkyl, aryl, heteroarylrespectively

“Sulfoxyl” refers to a —S(═O)₂OH radical.

“Sulfonate” refers to a —S(═O)₂—OR radical, where R is selected from thegroup consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded througha ring carbon) and heteroalicyclic (bonded through a ring carbon). Asulfonate group is optionally substituted on R by one or more of thesubstituents described for alkyl, cycloalkyl, aryl, heteroarylrespectively.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

Compounds of the present invention also include crystalline andamorphous forms of those compounds, including, for example, polymorphs,pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (includinganhydrates), conformational polymorphs, and amorphous forms of thecompounds, as well as mixtures thereof. “Crystalline form,” “polymorph,”and “novel form” may be used interchangeably herein, and are meant toinclude all crystalline and amorphous forms of the compound, including,for example, polymorphs, pseudopolymorphs, solvates, hydrates,unsolvated polymorphs (including anhydrates), conformational polymorphs,and amorphous forms, as well as mixtures thereof, unless a particularcrystalline or amorphous form is referred to.

Chemical entities include, but are not limited to, compounds of FormulaI, IV, V. VI, VII, VIII, IX, X, XI, XII, XIII, and XIV and allpharmaceutically acceptable forms thereof. Pharmaceutically acceptableforms of the compounds recited herein include pharmaceuticallyacceptable salts, chelates, non-covalent complexes, prodrugs, andmixtures thereof. In certain embodiments, the compounds described hereinare in the form of pharmaceutically acceptable salts. Hence, the terms“chemical entity” and “chemical entities” also encompasspharmaceutically acceptable salts, chelates, non-covalent complexes,prodrugs, and mixtures.

In addition, if the compound of Formula I, IV, V. VI, VII, VIII, IX, X,XI, XII, XIII, or XIV is obtained as an acid addition salt, the freebase can be obtained by basifying a solution of the acid salt.Conversely, if the product is a free base, an addition salt,particularly a pharmaceutically acceptable addition salt, may beproduced by dissolving the free base in a suitable organic solvent andtreating the solution with an acid, in accordance with conventionalprocedures for preparing acid addition salts from base compounds. Thoseskilled in the art will recognize various synthetic methodologies thatmay be used to prepare non-toxic pharmaceutically acceptable additionsalts.

In one aspect, the present invention provides a compound of Formula I ora pharmaceutically acceptable salt thereof:

wherein W_(a) ¹ is CR³ or N; W_(a) ² is CR⁵ or N; W_(a) ³ is CR⁶ or N;W_(a) ⁴ is N or CR⁷; W_(b) ⁵ is CR⁸, CHR⁸, or N,

wherein no more than two adjacent ring atoms selected from W_(a) ¹,W_(a) ², W_(a) ³, W_(a) ⁴, and W_(b) ⁵ are heteroatoms;

W_(d) is heterocycloalkyl, aryl or heteroaryl;

B is alkyl, amino, heteroalkyl, cycloalkyl, heterocycloalkyl, or amoiety of Formula II;

wherein W_(c) is aryl, heteroaryl, heterocycloalkyl, or cycloalkyl, andq is an integer of 0, 1, 2, 3, or 4;

X is absent or is —(CH(R⁹))z and each instance of z independently is aninteger of 1, 2, 3, or 4;

Y is absent, —O—, —S—, —S(═O)—, —S(═O)₂—, —N(R⁹)—, —C(═O)—(CHR⁹)_(z)—,—C(═O)—, —N(R⁹)—C(═O)—, or —N(R⁹)—C(═O)NH—, —N(R⁹)C(R⁹)₂—, or—C(═O)—(CHR⁹)_(z)—;

R¹ is hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, alkoxy,amido, amino, acyl, acyloxy, alkoxycarbonyl, sulfonamido, halo, cyano,hydroxy, nitro, phosphate, urea, or carbonate;

R² is alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, alkoxy,amido, amino, acyl, acyloxy, alkoxycarbonyl, sulfonamido, halo, cyano,hydroxy, nitro, phosphate, urea, or carbonate;

R³ is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl, sulfonamido, halo,cyano, hydroxy, nitro, aryl, or heteroaryl;

R⁵, R⁶, R⁷, and R⁸ are independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, heteroalkyl, alkoxy, amido, amino, acyl, acyloxy,sulfonamido, halo, cyano, hydroxy or nitro; and

each instance of R⁹ is independently hydrogen, C₁-C₁₀alkyl,C₃-C₇cycloalkyl, heterocycloalkyl, or C₂-C₁₀heteroalkyl.

In some embodiments, W_(a) ¹ is CR³. In some embodiments, W_(a) ¹ is N.In some embodiments, W_(a) ² is CR⁵. In some embodiments, W_(a) ² is N.In some embodiments, W_(a) ³ is CR⁶. In some embodiments, W_(a) ³ is N.In some embodiments, W_(a) ⁴ is CR⁷. In some embodiments, W_(a) ⁴ is N.In some embodiments, W_(b) ⁵ is CR⁸. In some embodiments, W_(b) ⁵ isCHR⁸. In some embodiments, W_(b) ⁵ is N. In some embodiments, W_(a) ² isCR⁵, W_(a) ³ is CR⁶ and W_(a) ⁴ is CR⁷. In some embodiments, W_(a) ² isN, W_(a) ³ is CR⁶, and W_(a) ⁴ is CR⁷. In some embodiments, W_(a) ² isCR⁵, W_(a) ³ is N, and W_(a) ⁴ is CR⁷. In some embodiments, W_(a) ² isCR⁵, W_(a) ³ is CR⁶, and W_(a) ⁴ is N. In some embodiments, W_(a) ² andW_(a) ³ are N and W_(a) ⁴ is CR⁷. In some embodiments, W_(a) ² is CR⁵,and W_(a) ³ and W_(a) ⁴ are N. In some embodiments, W_(b) ⁵ is CR⁸. Insome embodiments, W_(b) ⁵ is CHR⁸. In some embodiments, W_(b) ⁵ is N. Insome embodiments, W_(a) ² is CR⁵, W_(a) ³ is CR⁶, W_(a) ⁴ is CR⁷, andW_(b) ⁵ is CR⁸. In some embodiments, W_(a) ² is CR⁵, W_(a) ³ is CR⁶,W_(a) ⁴ is CR⁷, and W_(b) ⁵ is CHR⁸. In some embodiments, W_(a) ² isCR⁵, W_(a) ³ is CR⁶, W_(a) ⁴ is CR⁷, and W_(b) ⁵ is N. In someembodiments, W_(a) ² is N, W_(a) ³ is CR⁶, W_(a) ⁴ is CR⁷, and W_(b) ⁵is CR⁸. In some embodiments, W_(a) ² is N, W_(a) ³ is CR⁶, W_(a) ⁴ isCR⁷, and W_(b) ⁵ is CHR⁸. In some embodiments, W_(a) ² is N, W_(a) ³ isCR⁶, W_(a) ⁴ is CR⁷, and W_(b) ⁵ is N. In some embodiments, W_(a) ² isCR⁵, W_(a) ³ is N, W_(a) ⁴ is CR⁷, and W_(b) ⁵ is CR⁸. In someembodiments, W_(a) ² is CR⁵, W_(a) ³ is N, W_(a) ⁴ is CR⁷, and W_(b) ⁵is CHR⁸. In some embodiments, W_(a) ² is CR⁵, W_(a) ³ is N, W_(a) ⁴ isCR⁷, and W_(b) ⁵ is N. In some embodiments, W_(a) ² is CR⁵, W_(a) ³ isCR⁶, W_(a) ⁴ is N, and W_(b) ⁵ is CR⁸. In some embodiments, W_(a) ² isCR⁵, W_(a) ³ is CR⁶, W_(a) ⁴ is N, and W_(b) ⁵ is CHR⁸. In someembodiments, W_(a) ² is CR⁵, W_(a) ³ is CR⁶, W_(a) ⁴ is N, and W_(b) ⁵is N. In some embodiments, W_(a) ² and W_(a) ³ are N, W_(a) ⁴ is CR⁷,and W_(b) ⁵ is CR⁸. In some embodiments, W_(a) ² and W_(a) ³ are N,W_(a) ⁴ is CR⁷, and W_(b) ⁵ is CHR⁸. In some embodiments, W_(a) ² andW_(a) ³ are N, W_(a) ⁴ is CR⁷, and W_(b) ⁵ is N. In some embodiments,W_(a) ² is CR⁵, W_(a) ³ and W_(a) ⁴ are N, and W_(b) ⁵ is CR⁸. In someembodiments, W_(a) ² is CR⁵, W_(a) ³ and W_(a) ⁴ are N, and W_(b) ⁵ isCHR⁸. In some embodiments, W_(a) ² is CR⁵, W_(a) ³ and W_(a) ⁴ are N,and W_(b) ⁵ is N.

In some embodiments, B is unsubstituted or substituted alkyl, includingbut not limited to —(CH₂)₂—NR^(a)R^(a), wherein each R^(a) isindependently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, or NR^(a)R^(a) arecombined together to form a cyclic moiety, which includes but is notlimited to piperidinyl, piperazinyl, and morpholinyl. In someembodiments, B is unsubstituted or substituted amino. In someembodiments, B is unsubstituted or substituted heteroalkyl.

In some embodiments, B is a moiety of Formula II and wherein W_(c) is amember selected from the group consisting of unsubstituted orsubstituted aryl, substituted phenyl, unsubstituted or substitutedheteroaryl including but not limited to pyridin-2-yl, pyridin-3-yl,pyridin-4-yl, pyrimidin-4-yl, pyrimidin-2-yl, pyrimidin-5-yl, orpyrazin-2-yl, unsubstituted or substituted monocyclic heteroaryl,unsubstituted or substituted bicyclic heteroaryl, a heteroarylcomprising two heteroatoms as ring atoms, unsubstituted or substitutedheteroaryl comprising a nitrogen ring atom, heteroaryl comprising twonitrogen ring atoms, heteroaryl comprising a nitrogen and a sulfur asring atoms, unsubstituted or substituted heterocycloalkyl including butnot limited to morpholinyl, tetrahydropyranyl, piperazinyl, andpiperidinyl, unsubstituted or substituted cycloalkyl including but notlimited to cyclopentyl and cyclohexyl.

In some embodiments, B is one of the following moieties:

In some embodiments, B is substituted by one or more of alkyl,heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl,sulfonamido, halo, cyano, hydroxy or nitro, each of which alkyl,heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, alkoxy, amido, amino, acyl, acyloxy, or sulfonamido, mayitself be substituted.

In some embodiments, R¹ is a member selected from the group consistingof hydrogen, unsubstituted or substituted alkyl, unsubstituted orsubstituted heteroalkyl, unsubstituted or substituted alkenyl,unsubstituted or substituted alkynyl, unsubstituted or substitutedcycloalkyl, or unsubstituted or substituted heterocycloalkyl. In someembodiments, R¹ is unsubstituted or substituted aryl, unsubstituted orsubstituted arylalkyl, unsubstituted or substituted heteroaryl, orunsubstituted or substituted heteroarylalkyl. In some embodiments, R¹ isunsubstituted or substituted alkoxy, unsubstituted or substituted amido,unsubstituted or substituted amino. In some embodiments, R¹ isunsubstituted or substituted acyl, unsubstituted or substituted acyloxy,unsubstituted or substituted alkoxycarbonyl, or unsubstituted orsubstituted sulfonamido. In some embodiments, R¹ is halo which includes—Cl, —F, —I, and —Br. In some embodiments, R¹ is selected from the groupconsisting of cyano, hydroxy, nitro, unsubstituted or substitutedphosphate, unsubstituted or substituted urea, and carbonate.

In some embodiments, when R¹ is alkyl, R¹ is methyl, ethyl, propyl,isopropyl, n-butyl, tert-butyl, sec-butyl, pentyl, hexyl or heptyl.

In some embodiments, when R¹ is alkyl, heteroalkyl, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl,sulfonamido, or hydroxy, R¹ is substituted by phosphate, orunsubstituted urea, or substituted urea, or carbonic acid, or carbonate.

In some embodiments, when R¹ is alkyl, heteroalkyl, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl, orsulfonamido, R¹ is substituted by one or more of alkyl, heteroalkyl,alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl,heteroaryl, heteroarylalkyl, alkoxy, amido, amino, acyl, acyloxy,alkoxycarbonyl, sulfonamido, halo, cyano, hydroxy or nitro, each ofwhich alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, alkoxy,amido, amino, acyl, acyloxy, alkoxycarbonyl, or sulfonamido may itselfbe substituted.

In some embodiments, R² is a member selected from the group consistingof unsubstituted or substituted alkyl, unsubstituted or substitutedheteroalkyl, unsubstituted or substituted alkenyl, unsubstituted orsubstituted alkynyl, unsubstituted or substituted cycloalkyl, andunsubstituted or substituted heterocycloalkyl. In some embodiments, R²is unsubstituted or substituted aryl, unsubstituted or substitutedarylalkyl, unsubstituted or substituted heteroaryl, or unsubstituted orsubstituted heteroarylalkyl. In some embodiments, R² is unsubstituted orsubstituted alkoxy, unsubstituted or substituted amido, unsubstituted orsubstituted amino. In some embodiments, R² is unsubstituted orsubstituted acyl, unsubstituted or substituted acyloxy, unsubstituted orsubstituted alkoxycarbonyl, or unsubstituted or substituted sulfonamido.In some embodiments, R² is halo, which is —I, —F, —Cl, or —Br. In someembodiments, R² is selected from the group consisting of cyano, hydroxy,nitro, a carbonic acid, and a carbonate. In some embodiments, R² isunsubstituted or substituted phosphate. In some embodiments, R² isunsubstituted or substituted urea. In some embodiments, when R² isalkyl, R² is methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl,sec-butyl, pentyl, hexyl or heptyl.

In some embodiments, when R² is alkyl, heteroalkyl, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl,sulfonamido, or hydroxy, it is substituted by phosphate, substituted byurea, or substituted by carbonate.

In some embodiments, when R² is alkyl, heteroalkyl, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl, orsulfonamido, it is substituted by one or more of alkyl, heteroalkyl,alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl, sulfonamido, halo,cyano, hydroxy or nitro, each of which alkyl, heteroalkyl, alkenyl,alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, amido,amino, acyl, acyloxy, alkoxycarbonyl, or sulfonamido may itself besubstituted.

In some embodiments, q is an integer of 0. In some embodiments, q is aninteger of 1. In some embodiments, q is an integer of 2. In someembodiments, q is an integer of 3. In some embodiments, q is an integerof 4.

In some embodiments, R³ is a member selected from the group consistingof hydrogen, unsubstituted or substituted alkyl, unsubstituted orsubstituted alkenyl, and unsubstituted or substituted alkynyl. In someembodiments, R³ is unsubstituted or substituted aryl, unsubstituted orsubstituted heteroaryl, unsubstituted or substituted cycloalkyl, orunsubstituted or substituted heterocycloalkyl. In some embodiments, R³is unsubstituted or substituted alkoxy, unsubstituted or substitutedamido, unsubstituted or substituted amino. In some embodiments, R³ isunsubstituted or substituted acyl, unsubstituted or substituted acyloxy,unsubstituted or substituted alkoxycarbonyl, or unsubstituted orsubstituted sulfonamido. In some embodiments, R³ is halo, which is —I,—F, —Cl, or —Br.

In some embodiments, R³ is selected from the group consisting of cyano,hydroxy, and nitro. In some embodiments, when R³ is alkyl, R³ is methyl,ethyl, propyl, isopropyl, n-butyl, tert-butyl, sec-butyl, pentyl, hexylor heptyl. In some embodiments, R³ is —CF₃.

In some embodiments, when R³ is alkyl, alkenyl, alkynyl, aryl,heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, amido, amino, acyl,acyloxy, alkoxycarbonyl, or sulfonamido, it is substituted with one ormore of alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, alkoxy, amido, amino, acyl, acyloxy,alkoxycarbonyl, sulfonamido, halo, cyano, hydroxy or nitro, each ofwhich alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, alkoxy, amido, amino, acyl, acyloxy,alkoxycarbonyl, or sulfonamido may itself be substituted.

In some embodiments, R⁵ is hydrogen, unsubstituted or substituted alkyl(including but not limited to unsubstituted or substituted C₁-C₄alkyl).In some embodiments, R⁵ is unsubstituted or substituted alkenylincluding but not limited to unsubstituted or substituted C₂-C₅alkenyl.In some embodiments, R⁵ is unsubstituted or substituted alkynylincluding but not limited to unsubstituted or substituted C₂-C₅alkynyl.In some embodiments, R⁵ is unsubstituted or substituted cycloalkylincluding but not limited to unsubstituted or substitutedC₃-C₅cycloalkyl. In some embodiments, R⁵ is unsubstituted or substitutedheterocycloalkyl. In some embodiments, R⁵ is unsubstituted orsubstituted heteroalkyl including but not limited to unsubstituted orsubstituted C₁-C₄heteroalkyl. In some embodiments, R⁵ is unsubstitutedor substituted alkoxy including but not limited to unsubstituted orsubstituted C₁-C₄alkoxy. In some embodiments, R⁵ is unsubstituted orsubstituted amido including but not limited to unsubstituted orsubstituted C₁-C₄amido. In some embodiments, R⁵ is unsubstituted orsubstituted amino. In some embodiments, R⁵ is unsubstituted orsubstituted acyl, unsubstituted or substituted acyloxy, unsubstituted orsubstituted C₁-C₄acyloxy, unsubstituted or substituted alkoxycarbonyl,unsubstituted or substituted sulfonamido, or unsubstituted orsubstituted C₁-C₄sulfonamido. In some embodiments, R⁵ is halo, which is—I, —F, —Cl, or —Br. In some embodiments, R⁵ is selected from the groupconsisting of cyano, hydroxy, and nitro. In some other embodiments, R⁵is —CH₃, —CH₂CH₃, n-propyl, isopropyl, —OCH₃, —OCH₂CH₃, or —CF₃.

In some embodiments, when R⁵ is alkyl, alkenyl, alkynyl, cycloalkyl,heteroalkyl, acyl, alkoxy, amido, amino, acyloxy, alkoxycarbonyl, orsulfonamido, R⁵ is optionally substituted with one or more of alkyl,heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl,sulfonamido, halo, cyano, hydroxy or nitro, each of which alkyl,heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl, orsulfonamido may itself be substituted.

In some embodiments, R⁶ is hydrogen, unsubstituted or substituted alkyl(including but not limited to unsubstituted or substituted C₁-C₄alkyl).In some embodiments, R⁶ is unsubstituted or substituted alkenylincluding but not limited to unsubstituted or substituted C₂-C₅alkenyl.In some embodiments, R⁶ is unsubstituted or substituted alkynylincluding but not limited to unsubstituted or substituted C₂-C₅alkynyl.In some embodiments, R⁶ is unsubstituted or substituted cycloalkylincluding but not limited to unsubstituted or substitutedC₃-C₅cycloalkyl. In some embodiments, R⁶ is unsubstituted or substitutedheterocycloalkyl. In some embodiments, R⁶ is unsubstituted orsubstituted heteroalkyl including but not limited to unsubstituted orsubstituted C₁-C₄heteroalkyl. In some embodiments, R⁶ is unsubstitutedor substituted alkoxy including but not limited to unsubstituted orsubstituted C₁-C₄alkoxy. In some embodiments, R⁶ is unsubstituted orsubstituted amido including but not limited to unsubstituted orsubstituted C₁-C₄amido. In some embodiments, R⁶ is unsubstituted orsubstituted amino. In some embodiments, R⁶ is unsubstituted orsubstituted acyl, unsubstituted or substituted acyloxy, unsubstituted orsubstituted C₁-C₄acyloxy, unsubstituted or substituted alkoxycarbonyl,unsubstituted or substituted sulfonamido, or unsubstituted orsubstituted C₁-C₄sulfonamido. In some embodiments, R⁶ is halo, which is—I, —F, —Cl, or —Br. In some embodiments, R⁶ is selected from the groupconsisting of cyano, hydroxy, and nitro. In some other embodiments, R⁶is —CH₃, —CH₂CH₃, n-propyl, isopropyl, —OCH₃, —OCH₂CH₃, or —CF₃.

In some embodiments, when R⁶ is alkyl, alkenyl, alkynyl, cycloalkyl,heteroalkyl, acyl, alkoxy, amido, amino, acyloxy, alkoxycarbonyl, orsulfonamido, R⁶ is optionally substituted with one or more of alkyl,heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl,sulfonamido, halo, cyano, hydroxy or nitro, each of which alkyl,heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl, orsulfonamido may itself be substituted.

In some embodiments, R⁷ is hydrogen, unsubstituted or substituted alkyl(including but not limited to unsubstituted or substituted C₁-C₄alkyl).In some embodiments, R⁷ is unsubstituted or substituted alkenylincluding but not limited to unsubstituted or substituted C₂-C₅alkenyl.In some embodiments, R⁷ is unsubstituted or substituted alkynylincluding but not limited to unsubstituted or substituted C₂-C₅alkynyl.In some embodiments, R⁷ is unsubstituted or substituted cycloalkylincluding but not limited to unsubstituted or substitutedC₃-C₅cycloalkyl. In some embodiments, R⁷ is unsubstituted or substitutedheterocycloalkyl. In some embodiments, R⁷ is unsubstituted orsubstituted heteroalkyl including but not limited to unsubstituted orsubstituted C₁-C₄heteroalkyl. In some embodiments, R⁷ is unsubstitutedor substituted alkoxy including but not limited to unsubstituted orsubstituted C₁-C₄alkoxy. In some embodiments, R⁷ is unsubstituted orsubstituted amido including but not limited to unsubstituted orsubstituted C₁-C₄amido. In some embodiments, R⁷ is unsubstituted orsubstituted amino. In some embodiments, R⁷ is unsubstituted orsubstituted acyl, unsubstituted or substituted acyloxy, unsubstituted orsubstituted C₁-C₄acyloxy, unsubstituted or substituted alkoxycarbonyl,unsubstituted or substituted sulfonamido, or unsubstituted orsubstituted C₁-C₄sulfonamido. In some embodiments, R⁷ is halo, which is—I, —F, —Cl, or —Br. In some embodiments, R⁷ is selected from the groupconsisting of cyano, hydroxy, and nitro. In some other embodiments, R⁷is —CH₃, —CH₂CH₃, n-propyl, isopropyl, —OCH₃, —OCH₂CH₃, or —CF₃.

In some embodiments, when R⁷ is alkyl, alkenyl, alkynyl, cycloalkyl,heteroalkyl, acyl, alkoxy, amido, amino, acyloxy, alkoxycarbonyl, orsulfonamido, R⁷ is optionally substituted with one or more of alkyl,heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl,sulfonamido, halo, cyano, hydroxy or nitro, each of which alkyl,heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl, orsulfonamido may itself be substituted.

In some embodiments, R⁸ is hydrogen, unsubstituted or substituted alkyl(including but not limited to unsubstituted or substituted C₁-C₄alkyl).In some embodiments, R⁸ is unsubstituted or substituted alkenylincluding but not limited to unsubstituted or substituted C₂-C₅alkenyl.In some embodiments, R⁸ is unsubstituted or substituted alkynylincluding but not limited to unsubstituted or substituted C₂-C₅alkynyl.In some embodiments, R⁸ is unsubstituted or substituted cycloalkylincluding but not limited to unsubstituted or substitutedC₃-C₅cycloalkyl. In some embodiments, R⁸ is unsubstituted or substitutedheterocycloalkyl. In some embodiments, R⁸ is unsubstituted orsubstituted heteroalkyl including but not limited to unsubstituted orsubstituted C₁-C₄heteroalkyl. In some embodiments, R⁸ is unsubstitutedor substituted alkoxy including but not limited to unsubstituted orsubstituted C₁-C₄alkoxy. In some embodiments, R⁸ is unsubstituted orsubstituted amido including but not limited to unsubstituted orsubstituted C₁-C₄amido. In some embodiments, R⁸ is unsubstituted orsubstituted amino. In some embodiments, R⁸ is unsubstituted orsubstituted acyl, unsubstituted or substituted acyloxy, unsubstituted orsubstituted C₁-C₄acyloxy, unsubstituted or substituted alkoxycarbonyl,unsubstituted or substituted sulfonamido, or unsubstituted orsubstituted C₁-C₄sulfonamido. In some embodiments, R⁸ is halo, which is—I, —F, —Cl, or —Br. In some embodiments, R⁸ is selected from the groupconsisting of cyano, hydroxy, and nitro. In some other embodiments, R⁸is —CH₃, —CH₂CH₃, n-propyl, isopropyl, —OCH₃, —OCH₂CH₃, or —CF₃.

In some embodiments, when R⁸ is alkyl, alkenyl, alkynyl, cycloalkyl,heteroalkyl, acyl, alkoxy, amido, amino, acyloxy, alkoxycarbonyl, orsulfonamido, R⁸ is optionally substituted with one or more of alkyl,heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl,sulfonamido, halo, cyano, hydroxy or nitro, each of which alkyl,heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl, orsulfonamido may itself be substituted.

In some embodiments, R⁵, R⁶, R⁷, and R⁸ are H.

In some embodiments, X is absent. In some embodiments, X is—(CH(R⁹))_(z), and z is an integer of 1, 2, 3 or 4.

In some embodiments, R⁹ is unsubstituted or substituted alkyl includingbut not limited to unsubstituted or substituted C₁-C₁₀alkyl. In someembodiments, R⁹ is unsubstituted or substituted cycloalkyl including butnot limited to unsubstituted or substituted C₃-C₇cycloalkyl. In someembodiments, R⁹ is methyl or hydrogen. In some embodiments, R⁹ isunsubstituted or substituted heterocycloalkyl including but not limitedto unsubstituted or substituted C₂-C₁₀heteroalkyl. In some embodiments,R⁹ is unsubstituted or substituted heteroalkyl including but not limitedto unsubstituted or substituted C₂-C₁₀heteroalkyl.

When R⁹ is any of the above, in some embodiments, X is —CH₂—, —CH₂CH₂—,—CH₂CH₂CH₂—, —CH(CH₃)—, or —CH(CH₂CH₃)—. In some embodiments, when X is—CH(CH₃)—, —CH(CH₃)— is in an (S)- or (R)-stereochemical configuration.

In some embodiments of the compound of Formula I, Y is absent. In someembodiments, Y is —O—, —S—, —S(═O)—, —S(═O)₂—, —C(═O)—, —N(R⁹)(C═O)—,—N(R⁹)(C═O)NH—, —N(R⁹)C(R⁹)₂— (such as —N(R⁹)CH₂—, specifically—N(CH₃)CH₂—, N(CH(CH₃)₂)CH₂— or N(CH₂CH₃)CH₂—), —N(R⁹)—, —N(CH₃)—,—N(CH₂CH₃)—, or —N(CH(CH₃)₂)—. In some embodiments, Y is—C(═O)—(CHR⁹)_(z)— and z is an integer of 1, 2, 3, or 4.

In some embodiments, X—Y is —CH₂—, —CH₂—N(CH₃), —CH(CH₃)—NH—, (S)—CH(CH₃)—NH—, or (R) —CH(CH₃)—NH—. In some embodiments, X—Y is—N(CH₃)⁻CH₂—, N(CH₂CH₃) CH₂—, —N(CH(CH₃)₂)CH₂—, or —NHCH₂—.

In some embodiments, W_(d) is a member selected from the groupconsisting of unsubstituted or substituted heterocycloalkyl,unsubstituted or substituted aryl, and unsubstituted or substitutedheteroaryl. In some embodiments, W_(d) is unsubstituted or substitutedmonocyclic heteroaryl, or unsubstituted or substituted bicyclicheteroaryl. In some embodiments, W_(d) is a bicyclic heteroaryl havingat least one heteroatom, e.g., a bicyclic heteroaryl having at least onenitrogen ring atom. In some embodiments, W_(d) is a bicyclic heteroarylhaving at least two heteroatoms, e.g., a bicyclic heteroaryl having atleast two nitrogen ring atoms. In some embodiments, W_(d) is a bicyclicheteroaryl having two heteroatoms in the ring which is connected to XY.In some embodiments, W_(d) is a bicyclic heteroaryl having two nitrogenring atoms in the ring to which XY is connected. In some embodiments,W_(d) is a bicyclic heteroaryl having four heteroatoms, e.g., a bicyclicheteroaryl having four nitrogen ring atoms. In some embodiments, W_(d)is unsubstituted or substituted4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl, unsubstituted or substituted7-amino-2-methyl-2H-pyrazolo[4,3-d]pyrimidin-3-yl. unsubstituted orsubstituted 6-methylenyl-9H-purin-6-yl, or unsubstituted or substituted6-amino-9H-purin-9-yl.

In some embodiments W_(d) is one of the following:

wherein R^(a) is hydrogen, halo, phosphate, urea, carbonate, alkyl,alkenyl, alkynyl, cycloalkyl, heteroalkyl, or heterocycloalkyl;R¹¹ is H, alkyl, halo, amino, amido, hydroxy, or alkoxy, andR¹² is H, alkyl, cyano, alkynyl, alkenyl, halo, aryl, heteroaryl,heterocycloalkyl, cycloalkyl, amino, carboxylic acid, alkoxycarbonyl, oramido.

In some embodiments, W_(d) is:

In some embodiments, W_(d) is:

In some embodiments, W_(d) is:

In some embodiments, W_(d) is:

In some embodiments of W_(d), R^(a) is a member selected from the groupconsisting of hydrogen, halo, phosphate, urea, a carbonate,unsubstituted or substituted alkyl, unsubstituted or substitutedalkenyl, unsubstituted or substituted alkynyl, unsubstituted orsubstituted cycloalkyl, unsubstituted or substituted heteroalkyl, andunsubstituted or substituted heterocycloalkyl.

In some embodiments of W_(d), when R^(a) is alkyl, alkynyl, cycloalkyl,heteroalkyl, or heterocycloalkyl, it is substituted by phosphate, urea,or carbonate.

In some embodiments, R¹¹ is a member of the group consisting ofhydrogen, unsubstituted or substituted alkyl, and halo, which includes—I, —F, —Cl, or —Br. In some embodiments, R¹¹ is unsubstituted orsubstituted amino, unsubstituted or substituted amido, hydroxy, orunsubstituted or substituted alkoxy. In some embodiments, R¹¹ isphosphate, unsubstituted or substituted urea, or carbonate.

In some embodiments, when R¹¹ is alkyl, amino, amido, hydroxy, oralkoxy, it is substituted by phosphate, urea, or carbonate.

In some embodiments, —X—Y—W_(d) is one of the following moieties:

In some embodiments, R¹² is a member of the group consisting ofhydrogen, cyano, halo, unsubstituted or substituted alkyl, andunsubstituted or substituted alkynyl unsubstituted or substitutedalkenyl. In some embodiments, R¹² is unsubstituted or substituted aryl.In some embodiments, R¹² is unsubstituted or substituted heteroaryl,which includes but is not limited to heteroaryl having a 5 memberedring, heteroaryl having a six membered ring, heteroaryl with at leastone nitrogen ring atom, heteroaryl with two nitrogen ring atoms,monocylic heteroaryl, and bicyclic heteroaryl. In some embodiments, R¹²is unsubstituted or substituted heterocycloalkyl, which includes but isnot limited to heterocycloalkyl with one nitrogen ring atom,heterocycloalkyl with one oxygen ring atom, R¹² is heterocycloalkyl withone sulfur ring atom, 5 membered heterocycloalkyl, 6 memberedheterocycloalkyl, saturated heterocycloalkyl, unsaturatedheterocycloalkyl, heterocycloalkyl having an unsaturated moietyconnected to the heterocycloalkyl ring, heterocycloalkyl substituted byoxo, and heterocycloalkyl substituted by two oxo. In some embodiments,R¹² is unsubstituted or substituted cycloalkyl, including but notlimited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkylsubstituted by one oxo, cycloalkyl having an unsaturated moietyconnected to the cycloalkyl ring. In some embodiments, R¹² isunsubstituted or substituted amido, carboxylic acid, unsubstituted orsubstituted acyloxy, or unsubstituted or substituted alkoxycarbonyl.

In some embodiments, when R¹² is alkyl, alkynyl, alkenyl, aryl,heteroaryl, heterocycloalkyl, or cycloalkyl, it is substituted withphosphate. In some embodiments, when R¹² is alkyl, alkynyl, alkenyl,aryl, heteroaryl, heterocycloalkyl, or cycloalkyl, it is substitutedwith urea. In some embodiments, when R¹² is alkyl, alkynyl, alkenyl,aryl, heteroaryl, heterocycloalkyl, or cycloalkyl, it is substitutedwith carbonate.

In some embodiments, when R¹² is alkyl, alkynyl, alkenyl, aryl,heteroaryl, heterocycloalkyl, cycloalkyl, alkoxycarbonyl, amido, oracyloxy, it is substituted with one or more of alkyl, heteroalkyl,alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl, sulfonamido, halo,cyano, hydroxy or nitro, each of which alkyl, heteroalkyl, alkenyl,alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, amido,amino, acyl, acyloxy, alkoxycarbonyl, or sulfonamido may itself besubstituted.

In some embodiments, R¹² of W_(d) is one of the following moieties:

In some embodiments, W_(d) is a pyrazolopyrimidine of Formula III:

wherein R¹¹ is H, alkyl, halo, amino, amido, hydroxy, or alkoxy, and R¹²is H, alkyl, alkynyl, alkenyl, halo, aryl, heteroaryl, heterocycloalkyl,or cycloalkyl. In some embodiments, R¹¹ is amino and R¹² is H, alkyl,alkynyl, alkenyl, halo, aryl, heteroaryl, heterocycloalkyl, orcycloalkyl. In some embodiments, R¹¹ is amino and R¹² is alkyl, halo,aryl, heteroaryl, heterocycloalkyl, or cycloalkyl. In some embodiments,R¹¹ is amino and R¹² is monocyclic heteroaryl. In some embodiments, R¹¹is amino and R¹² is bicyclic heteroaryl. In some embodiments, R¹¹ isamino and R¹² is cyano, amino, carboxylic acid, acyloxy, alkoxycarbonyl,or amido.

In some embodiments, the compound of Formula I is a compound which has astructure selected from the group consisting of Formula 1-A, 1-B, 2-A,2-B. IV, V, V-A, VI, VI-A, VI-B, VI-C, 6-C1, 6-C2, V-I-D, and 6-D:

In another embodiment, the compound of Formula I is a compound which hasa structure selected from the group consisting of Formula VII, 7-A,VIII, VIII-A, and 8-A:

Any of the disclosed elements and their substituents for the compoundsof Formula I can be used in any combination.

In one aspect, for compounds of Formula I, IV, V, VI, VII, or VIII, R₃is H, CH₃, CF₃, Cl, F, aryl, or heteroaryl; B is alkyl or a moiety ofFormula II;

wherein W_(c) is aryl, heteroaryl, heterocycloalkyl, or cycloalkyl; R¹is H, —F, —Cl, —CN, —CH₃, isopropyl, —CF₃, —OCH₃, nitro, or phosphate;R² is halo, hydroxy, cyano, nitro, or phosphate; q is an integer of 0,1, 2, 3, or 4; R⁵, R⁶, R⁷, and R⁸ are H; X is absent or (CH₂)_(z); z is1; Y is absent, —N(R⁹)—, or —N(R⁹) CH(R⁹)—; R⁹ is hydrogen, C₁-C₁₀alkyl,C₃-C₇cycloalkyl, or C₂-C₁₀heteroalkyl; and W_(d) is pyrazolopyrimidineor purine.

In another aspect, for compounds of Formula I, IV, V, VI, VII, or VIII,R₃ is H, CH₃, CF₃, Cl, or F; B is alkyl or a moiety of Formula II whichis aryl, heteroaryl, heterocycloalkyl, or cycloalkyl; R¹ is H, —F, —Cl,—CN, —CH₃, isopropyl, —CF₃, —OCH₃, nitro, or phosphate; R² is halo,hydroxy, cyano, nitro, or phosphate; q is 0, 1 or 2; R⁵, R⁶, R⁷, and R⁸are H; X is absent or (CH₂)_(z); z is 1; Y is absent, —N(R⁹)—, or —N(R⁹)CH(R⁹)—; R⁹ is hydrogen, methyl, or ethyl; W_(d) is:

R¹¹ is amino; and R¹² is H, alkyl, alkynyl, alkenyl, halo, aryl,heteroaryl, heterocycloalkyl, cycloalkyl, cyano, amino, carboxylic acid,alkoxycarbonyl, or amido.

In another aspect, for compounds of Formula I, IV, V, VI, VII, or VIII,R₃ is H, CH₃, CF₃, Cl, or F; B is alkyl or a moiety of Formula II whichis aryl, heteroaryl, heterocycloalkyl, or cycloalkyl; R¹ is H, —F, —Cl,—CN, —CH₃, isopropyl, —CF₃, —OCH₃, nitro, or phosphate; R² is halo,hydroxy, cyano, nitro or phosphate; q is 0, 1 or 2; R⁵, R⁶, R⁷, and R⁸are H; X is (CH₂)_(z); z is 1; Y is absent and W_(d) is:

R¹¹ is amino; and R¹² is H, alkyl, alkynyl, alkenyl, halo, aryl,heteroaryl, heterocycloalkyl, cycloalkyl, cyano, amino, carboxylic acid,alkoxycarbonyl, or amido.

In another aspect, for compounds of Formula I, IV, V, VI, VII, or VIII,R₃ is H, CH₃, CF₃, Cl, or F; B is alkyl or a moiety of Formula II, whichis aryl, heteroaryl, heterocycloalkyl, or cycloalkyl; R¹ is H, —F, —Cl,—CN, —CH₃, isopropyl, —CF₃, —OCH₃, nitro, or phosphate; R² is halo,hydroxy, cyano, nitro, or phosphate; q is 0, 1 or 2; R⁵, R⁶, R⁷, and R⁸are H; X is (CH₂)_(z); z is 1; Y is —N(R⁹)—; R⁹ is hydrogen, methyl, orethyl; and W_(d) is

In another aspect, for compounds of Formula I, IV, V, VI, VII, or VIII,R₃ is H, CH₃, CF₃, Cl, or F; B is alkyl or a moiety of Formula II, whichis aryl, heteroaryl, heterocycloalkyl, or cycloalkyl; R¹ is H, —F, —Cl,—CN, —CH₃, isopropyl, —CF₃, —OCH₃, nitro, or phosphate; R² is halo,hydroxy, cyano, nitro, or phosphate; q is 0, 1 or 2; R⁵, R⁶, R⁷, and R⁸are H; X is absent; Y is —N(R⁹) CH(R⁹)—; R⁹ is hydrogen, methyl, orethyl; and W_(d) is

The invention also provides a compound of Formula IX or itspharmaceutically acceptable salt,

wherein W_(a) ¹ and W_(a) ² are independently CR⁵, S, N, or NR⁴, andW_(a) ⁴ is independently CR⁷, S, N, or NR⁴ wherein no more than twoadjacent ring atoms are nitrogen or sulfur, and when W_(a) ¹ is S, oneof W_(a) ² and W_(a) ⁴ is N or NR⁴;

W_(b) ⁵ is CR⁸, N, or NR⁸;

B is alkyl, amino, heteroalkyl, cycloalkyl, heterocycloalkyl, or amoiety of Formula II;

wherein W_(c) is aryl, heteroaryl, heterocycloalkyl, or cycloalkyl, and

q is an integer of 0, 1, 2, 3, or 4;

W_(d) is absent or is a heterocycloalkyl, aryl or heteroaryl moiety;

X is absent or is —(CH(R⁹))_(z)— and each instance of z independently isan integer of 1, 2, 3, or 4;

Y is absent, —O—, —S—, —S(═O)—, —S(═O)₂—, —N(R⁹)—, —C(═O)—(CHR⁹)_(z)—,—C(═O)—, —N(R⁹)—C(═O)—, or —N(R⁹)—C(═O)NH—, —N(R⁹)C(R⁹)₂—, or—C(═O)—(CHR⁹)_(z)—;

R¹ is hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, alkoxy,amido, amino, acyl, acyloxy, alkoxycarbonyl, sulfonamido, halo, cyano,hydroxy, nitro, phosphate, urea, or carbonate;

R² is alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, alkoxy,amido, amino, acyl, acyloxy, alkoxycarbonyl, sulfonamido, halo, cyano,hydroxy, nitro, phosphate, urea, or carbonate;

R⁴ is hydrogen, acyl, alkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, or C₁-C₄heteroalkyl;

R⁵, R⁷, and R⁸ are independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, heteroalkyl, alkoxy, amido, amino, acyl, acyloxy,alkoxycarbonyl, sulfonamido, halo, cyano, hydroxy or nitro;

each instance of R⁹ is independently hydrogen, C₁-C₁₀alkyl,C₃-C₇cycloalkyl, heterocycloalkyl, or C₂-C₁₀heteroalkyl.

Substituents described for Formula I are equally applicable to compoundsof Formula IX, except for W_(a) ¹, W_(a) ², W_(a) ⁴, and W_(b) ⁵ whichare defined as follows:

In some embodiments, W_(a) ¹ is CR⁵, S, N, or NR⁴.

In some embodiments, W_(a) ² is CR⁵, S, N, or NR⁴.

In some embodiments, W_(a) ⁴ is CR⁷, S, N, or NR⁴.

In some embodiments, W_(b) ⁵ is CR⁸, N, or NR⁸.

In some embodiments, the compound of Formula IX has a structure which isa member of the group consisting of: (i) W_(a) ¹ is NR⁴, W_(a) ² is CR⁵,W_(a) ⁴ is CR⁷, and W_(b) ⁵ is CR⁸; (ii) W_(a) ¹ is NR⁴, W_(a) ² is CR⁵,W_(a) ⁴ is CR⁷, and W_(b) ⁵ is CHR⁸; (iii) W_(a) ¹ is NR⁴, W_(a) ² isCR⁵, W_(a) ⁴ is CR⁷, and W_(b) ⁵ is N; (iv) W_(a) ¹ is NR⁴, W_(a) ² isCR⁵, W_(a) ⁴ is CR⁷, and W_(b) ⁵ is NR⁸; (v) W_(a) ¹ is NR⁴, W_(a) ² isN, W_(a) ⁴ is CR⁷, and W_(b) ⁵ is CR⁸; (vi) W_(a) ¹ is NR⁴, W_(a) ² isN, W_(a) ⁴ is CR⁷, and W_(b) ⁵ is CHR⁸; (vii) W_(a) ¹ is NR⁴, W_(a) ² isN, W_(a) ⁴ is CR⁷, and W_(b) ⁵ is N; (viii) W_(a) ¹ is NR⁴, W_(a) ² isN, W_(a) ⁴ is CR⁷, and W_(b) ⁵ is NR⁸; (ix) W_(a) ¹ is NR⁴, W_(a) ² isCR⁵, W_(a) ⁴ is N, and W_(b) ⁵ is CR⁸; (x) W_(a) ¹ is NR⁴, W_(a) ² isCR⁵, W_(a) ⁴ is N, and W_(b) ⁵ is CHR⁸; (xi) W_(a) ¹ is NR⁴, W_(a) ² isCR⁵, W_(a) ⁴ is N, and W_(b) ⁵ is N; (xii) W_(a) ¹ is NR⁴, W_(a) ² isCR⁵, W_(a) ⁴ is N, and W_(b) ⁵ is NR⁸; (xiii) W_(a) ¹ is S, W_(a) ² isCR⁵, W_(a) ⁴ is N, and W_(b) ⁵ is CR⁸; (xiv) W_(a) ¹ is S, W_(a) ² isCR⁵, W_(a) ⁴ is N, and W_(b) ⁵ is CHR⁸; (xv) W_(a) is S, W_(a) ² is CR⁵,W_(a) ⁴ is N, and W_(b) ⁵ is N; (xvi) W_(a) ¹ is S, W_(a) ² is CR⁵,W_(a) ⁴ is N, and W_(b) ⁵ is NR⁸; (xvii) W_(a) ¹ is N, W_(a) ² is CR⁵,W_(a) ⁴ is S, and W_(b) ⁵ is CR⁸; (xviii) W_(a) ¹ is N, W_(a) ² is CR⁵,W_(a) ⁴ is S, and W_(b) ⁵ is CHR⁸; (xix) W_(a) ¹ is N, W_(a) ² is CR⁵,W_(a) ⁴ is S, and W_(b) ⁵ is N; (xx) W_(a) ¹ is N, W_(a) ² is CR⁵, W_(a)⁴ is S, and W_(b) ⁵ is NR⁸; (xxi) W_(a) ¹ is CR⁵, W_(a) ² is N, W_(a) ⁴is S, and W_(b) ⁵ is CR⁸; (xxi) W_(a) ¹ is CR⁵, W_(a) ² is N, W_(a) ⁴ isS, and W_(b) ⁵ is CHR⁸; (xxii) W_(a) ¹ is CR⁵, W_(a) ² is N, W_(a) ⁴ isS, and W_(b) ⁵ is N; (xxiii) W_(a) ¹ is CR⁵, W_(a) ² is N, W_(a) ⁴ is S,and W_(b) ⁵ is NR⁸; (xxiv) W_(a) ¹ is S, W_(a) ² is N, W_(a) ⁴ is CR⁷,and W_(b) ⁵ is CR⁸; (xxv) W_(a) ¹ is S, W_(a) ² is N, W_(a) ⁴ is CR⁷,and W_(b) ⁵ is CHR⁸; (xxvi) W_(a) ¹ is S, W_(a) ² is N, W_(a) ⁴ is CR⁷,and W_(b) ⁵ is N; (xxvii) W_(a) ¹ is S, W_(a) ² is N, W_(a) ⁴ is CR⁷,and W_(b) ⁵ is NR⁸; (xxviii) W_(a) ¹ is CR⁵, W_(a) ² is N, W_(a) ⁴ isNR⁴, and W_(b) ⁵ is CR⁸; (xxix) W_(a) ¹ is CR⁵, W_(a) ² is N, W_(a) ⁴ isNR⁴, and W_(b) ⁵ is CHR⁸; (xxx) W_(a) ¹ is CR⁵, W_(a) ² is N, W_(a) ⁴ isNR⁴, and W_(b) ⁵ is N; (xxxi) W_(a) ¹ is CR⁵, W_(a) ² is N, W_(a) ⁴ isNR⁴, and W_(b) ⁵ is NR⁸; (xxxii) W_(a) ¹ is CR⁵, W_(a) ² is CR⁵, W_(a) ⁴is S, and W_(b) ⁵ is CHR⁸; (xxxiii) W_(a) ¹ is CR⁵, W_(a) ² is CR⁵,W_(a) ⁴ is S, and W_(b) ⁵ is CR⁸; (xxxiv) W_(a) ¹ is CR⁵, W_(a) ² isCR⁵, W_(a) ⁴ is S, and W_(b) ⁵ is N; and (xxxv) W_(a) ¹ is CR⁵, W_(a) ²is CR⁵, W_(a) ⁴ is S, and W_(b) ⁵ is NR⁸.

In some embodiments, R⁴ is a member of the group consisting of hydrogen,unsubstituted or substituted acyl, unsubstituted or substituted alkylwhich includes but is not limited to unsubstituted or substitutedC₁-C₄alkyl, unsubstituted or substituted alkenyl which includes but isnot limited to C₂-C₅alkenyl, unsubstituted or substituted alkynyl whichincludes but is not limited to C₂-C₅alkynyl, unsubstituted orsubstituted cycloalkyl which includes but is not limited toC₃-C₅cycloalkyl, unsubstituted or substituted heterocycloalkyl, andunsubstituted or substituted heteroalkyl which includes but is notlimited to unsubstituted or substituted C₁-C₄heteroalkyl.

In some embodiments, when R⁴ is acyl, alkyl, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, or heteroalkyl, it is substituted with oneor more of alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, alkoxy, amido, amino, acyl, acyloxy,sulfonamido, halo, cyano, hydroxy or nitro, each of which alkyl,heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, alkoxy, amido, amino, acyl, acyloxy, or sulfonamido mayitself be substituted.

In some embodiments, the compound of Formula IX is a compound which hasa structure selected from the group consisting of Formula X, XI, XII,XIII, and XIV:

Any of the disclosed elements and their substituents for the compoundsof Formula IX can be used in any combination.

In one aspect, for compounds of Formula IX, X, XI, XII, XIII or XIV, Bis alkyl or a moiety of Formula II;

wherein W_(c) is aryl, heteroaryl, heterocycloalkyl, or cycloalkyl; R¹is H, —F, —Cl, —CN, —CH₃, isopropyl, —CF₃, —OCH₃, nitro, or phosphate;R² is halo, hydroxy, cyano, nitro, or phosphate; q is an integer of 0,1, 2, 3, or 4; R⁴, R⁵, R⁷, and R⁸ are H or methyl; X is absent or(CH₂)_(z); z is 1; Y is absent, —N(R⁹)—, or —N(R⁹) CH(R⁹)—; R⁹ ishydrogen, C₁-C₁₀alkyl, C₃-C₇cycloalkyl, or C₂-C₁₀heteroalkyl; and W_(d)is pyrazolopyrimidine or purine.

In another aspect, for compounds of Formula IX, X, XI, XII, XIII or XIV,B is alkyl or a moiety of Formula II wherein W_(c) is aryl, heteroaryl,heterocycloalkyl, or cycloalkyl; R¹ is H, —F, —Cl, —CN, —CH₃, isopropyl,—CF₃, —OCH₃, nitro, or phosphate; R² is halo, hydroxy, cyano, nitro, orphosphate; q is an integer of 0, 1, 2, 3, or 4; R⁴, R⁵, and R⁷ are H ormethyl; R⁸ is H; X is absent or (CH₂)_(z); z is 1; Y is absent or—N(R⁹)—; R⁹ is hydrogen, methyl, or ethyl; W_(d) is:

R¹¹ is amino; and R¹² is H, alkyl, alkynyl, alkenyl, halo, aryl,heteroaryl, heterocycloalkyl, cycloalkyl, cyano, amino, carboxylic acid,alkoxycarbonyl, or amido.

In another aspect, for compounds of Formula IX, X, XI, XII, XIII or XIV,B is a moiety of Formula II wherein W_(c) is aryl, heteroaryl,heterocycloalkyl, or cycloalkyl; R¹ is H, —F, —Cl, —CN, —CH₃, isopropyl,—CF₃, —OCH₃, or nitro; R² is halo, hydroxy, cyano, or nitro; q is 0, 1or 2; R⁴, R⁵, and R⁷ are H or methyl; R⁸ is H; X is (CH₂)_(z); z is 1; Yis absent and W_(d) is:

R¹¹ is amino; and R¹² is H, alkyl, alkynyl, alkenyl, halo, aryl,heteroaryl, heterocycloalkyl, or cycloalkyl.

In another aspect, for compounds of Formula IX, X, XI, XII, XIII or XIV,B is alkyl or a moiety of Formula II wherein W_(c) is aryl, heteroaryl,heterocycloalkyl, or cycloalkyl, R¹ is H, —F, —Cl, —CN, —CH₃, isopropyl,—CF₃, —OCH₃, nitro, or phosphate; R² is halo, hydroxy, cyano, nitro, orphosphate; q is 0, 1 or 2; R⁴, R⁵, and R⁷ are H or methyl; R⁸ is H; X is(CH₂)_(z); z is 1; Y is absent and W_(d) is:

R¹¹ is amino; and R¹² is H, alkyl, alkynyl, alkenyl, halo, aryl,heteroaryl, heterocycloalkyl, cycloalkyl, cyano, amino, carboxylic acid,alkoxycarbonyl, or amido.

In another aspect, for compounds of Formula IX, X, XI, XII, XIII or XIV,B is alkyl or a moiety of Formula II wherein W_(c) is aryl, heteroaryl,heterocycloalkyl, or cycloalkyl; R¹ is H, —F, —Cl, —CN, —CH₃, isopropyl,—CF₃, —OCH₃, nitro, or phosphate; R² is halo, hydroxy, cyano, nitro, orphosphate; q is 0, 1 or 2; R⁴, R⁵, and R⁷ are H or methyl; R⁸ is H; X is(CH₂)_(z); z is 1; X is (CH₂)_(z); z is 1; Y is —N(R⁹)—; R⁹ is hydrogen,methyl, or ethyl; and W_(d) is

In another aspect, for compounds of Formula IX, X, XI, XII, XIII or XIV,B is alkyl or a moiety of Formula II wherein W_(c) is aryl, heteroaryl,heterocycloalkyl, or cycloalkyl, R¹ is H, —F, —Cl, —CN, —CH₃, isopropyl,—CF₃, —OCH₃, nitro, or phosphate; R² is halo, hydroxy, cyano, nitro, orphosphate; q is 0, 1 or 2; R⁴, R⁵, and R⁷ are H or methyl; R⁸ is H; X isabsent; Y is —N(R⁹) CH(R⁹)—; R⁹ is hydrogen, methyl, or ethyl; W_(d) is:

Illustrative embodiments of Formula 1-A and Formula 1-B are provided,wherein R³ is selected from any of H, Cl, F, or methyl; any of W_(a) ²is elected from CH, N, C—CN, or C—OCH₃; any of W_(a) ³ is selected fromCH, N, C—CF₃, or C—CH₃; any of W_(a) ⁴ is selected from CH, N, or C—CF₃;any of R⁸ is selected from H, Me, or Cl; any of B as described in Table1; and any of R¹² as described in Table 2. The compounds of Formula 1-Aand Formula 1-B can contain any substituents specified under R³, W_(a)², W_(a) ³, W_(a) ⁴, R⁸, B, and R¹². The specific embodiments describedin no way limit the invention, but are descriptive of the compounds ofthe invention. Some additional exemplary compounds of Formulas 1-A and1-B are illustrated in Table 5.

TABLE 1 Illustrative B of a compound having a structure of Formula I,IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV. Sub- class # B B-1

B-2

B-3 —CH(CH₃)2 B-4

B-5

B-6

B-7

B-8

B-9

B-10

B-11

B-12

B-13

B-14

B-15

B-16

B-17

B-18

B-19

B-20

B-21

B-22

B-23

B-24

B-25

B-26

B-27

B-28

B-29

B-30

B-31

B-32

B-33

B-34

B-35

B-36

B-37

B-38

B-39

B-40

B-41

B-42

B-43

B-44

B-45

B-46

B-47

B-48

B-49

B-50

B-51

B-52

B-53

B-54

B-55

B-56

B-57

B-58

B-59

B-60

B-61

B-62

B-63

B-64

B-65

B-66

B-67

B-68

B-69

B-70

B-71

B-72

B-73

B-74

B-75

B-76

B-77

B-78

B-79

B-80

B-81

B-82

B-83

B-84

B-85

B-86

B-87 —CH₃ B-88 —CH₂CH₃ B-89

B-90

B-91

B-92

B-93

B-94

B-95

B-96

B-97

B-98

B-99

B-100

B-101

B-102

TABLE 2 Illustrative R¹² of a compound having a structure of Formula I,IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV. Sub- class # R¹²12-1 —CN 12-2 —Br 12-3 —Cl 12-4 —CH₂CH₃ 12-5 —CH₃ 12-6 —CH(CH₃)₂ 12-7

12-8

12-9

12-10

12-11

12-12

12-13

12-14

12-15

12-16

12-17

12-18

12-19

12-20

12-21

12-22

12-23

12-24

12-25

12-26

12-27

12-28

12-29

12-30

12-31

12-32

12-33

12-34

12-35 —H 12-36

12-37

12-38

12-39

12-40

12-41

12-42

12-43

12-44

12-45

12-46

12-47

12-48

12-49

12-50

12-51

12-52

12-53

12-54

12-55

12-56

12-57

12-58

12-59

12-60

12-61 —I 12-62

12-63

12-64

12-65

12-66

12-67

12-68

12-69

12-70

12-71

12-72

12-73

12-74

12-75

12-76

12-77

12-78

12-79

12-80

12-81

12-82

12-83

12-84

12-85

12-86

12-87

12-88

12-89

12-90

12-91

12-92

12-93

12-94

12-95

12-96

Illustrative embodiments of Formula 2-A and Formula 2-B are provided,wherein R³ is selected from any of H, Cl, F, or methyl; any of W_(a) ²is elected from CH, N, C—CN, or C—OCH₃; any of W_(a) ³ is selected fromCH, N, C—CF₃, or C—CH₃; any of W_(a) ⁴ is selected from CH, N, or C—CF₃;any of R⁸ is selected from H, Me, or Cl; any of B as described in Table1; any of R¹² as described in Table 2, and any of X—Y—W_(d) as describedin Table 3. The compounds of Formula 2-A and Formula 2-B can contain anysubstituents specified under R³, W_(a) ², W_(a) ³, W_(a) ⁴, R⁸, B, R¹²,and X—Y—W_(d). The specific embodiments described in no way limit theinvention, but are descriptive of the compounds of the invention. Someadditional exemplary compounds of Formulas 2-A and 2-B are illustratedin Table 5.

TABLE 3 Exemplary X—Y—W_(d) for a compound having a structure of FormulaI, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV. Sub- class #X—Y—W_(d) 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

Illustrative embodiments of Formula 6-A are provided, wherein R³ isselected from any of H, Cl, F, or methyl; any of B as described in Table1; and any of R¹² as described in Table 2. The compounds of Formula 6-Acan contain any substituents specified under R³, B, and R¹². Thespecific embodiments described in no way limit the invention, but aredescriptive of the compounds of the invention. Some additional exemplarycompounds of Formulas 6-A are illustrated in Table 5.

Illustrative embodiments of Formula 6-C1 are provided, wherein R³ isselected from any of H, Cl, F, or methyl; any of B as described in Table1; any of R⁹, which is selected from —H, —CH₃, or —CH₂CH₃; and any ofR¹² as described in Table 2. The compounds of Formula 6-C1 can containany substituents specified under R³, B, R⁹, and R¹². The specificembodiments described in no way limit the invention, but are descriptiveof the compounds of the invention. Some additional exemplary compoundsof Formulas 6-C1 are illustrated in Table 5.

Illustrative embodiments of Formula 6-C2 are provided, wherein R³ isselected from any of H, Cl, F, or methyl; any of B as described in Table1; and any of R⁹, which is selected from —H, —CH₃, or —CH₂CH₃. Thecompounds of Formula 6-C2 can contain any substituents specified underR³, B, and R⁹. The specific embodiments described in no way limit theinvention, but are descriptive of the compounds of the invention. Someadditional exemplary compounds of Formulas 6-C2 are illustrated in Table5.

Illustrative embodiments of Formula 6-D are provided, wherein R³ isselected from any of H, Cl, F, or methyl; any of B as described in Table1; and any of R⁹, which is selected from —H, —CH₃, or —CH₂CH₃. Thecompounds of Formula 6-D can contain any substituents specified underR³, B, and R⁹. The specific embodiments described in no way limit theinvention, but are descriptive of the compounds of the invention. Someadditional exemplary compounds of Formulas 6-D are illustrated in Table5.

Illustrative embodiments of Formula VIII are provided, wherein R³ isselected from any of H, Cl, F, or methyl; any of W_(a) ² is elected fromCH, N, C—CN, or C—OCH₃; any of W_(a) ³ is selected from CH, N, C—CF₃, orC—CH₃; any of W_(a) ⁴ is selected from CH, N, or C—CF₃; any of B asdescribed in Table 1; any of R¹² as described in Table 2, and any ofX—Y—W_(d) as described in Table 3. The compounds of Formula VIII cancontain any substituents specified under R³, W_(a) ², W_(a) ³, W_(a) ⁴,B, R¹², and X—Y—W_(d). The specific embodiments described in no waylimit the invention, but are descriptive of the compounds of theinvention. Some additional exemplary compounds of Formula VIII areillustrated in Table 5.

Also provided are compounds of substructure Formulae 9A-9BD undergeneric structure Formula IX, wherein R⁴ is selected from —H, methyl,ethyl, n-propyl, iso-propyl, cyclopropyl, cyclobutyl, and cyclopentyl;any of R⁵ is selected from H, Cl, F, methyl, or trifluoromethyl; any ofR⁷ is selected from H, Cl, F, methyl, trifluoromethyl; cyano, hydroxyl,ethyl, iso-propyl, and cyclopropyl; any of R⁸ is selected from H, methylor iso-propyl; any of B as described in Table 1; any of X—Y—W_(d) asdescribed in Table 3, and any of R¹² as described in Table 2. Thecompounds of Formulae 9A-9BD can contain any substituents specifiedunder R⁴, R⁵, R⁷ R⁸ B, X—Y—W_(d), and R¹². The specific embodimentsdescribed in no way limit the invention, but are descriptive of thecompounds of the invention. Some additional exemplary compounds ofFormulae 9-A to 9-BD are illustrated in Table 5.

Other illustrative compounds include but are not limited to thefollowing:

The chemical entities described herein can be synthesized according toone or more illustrative schemes herein and/or techniques well known inthe art.

Unless specified to the contrary, the reactions described herein takeplace at atmospheric pressure, generally within a temperature range from−10° C. to 200° C. Further, except as otherwise specified, reactiontimes and conditions are intended to be approximate, e.g., taking placeat about atmospheric pressure within a temperature range of about −10°C. to about 110° C. over a period of about 1 to about 24 hours;reactions left to run overnight average a period of about 16 hours.

The terms “solvent,” “organic solvent,” or “inert solvent” each mean asolvent inert under the conditions of the reaction being described inconjunction therewith including, for example, benzene, toluene,acetonitrile, tetrahydrofuran (“THF”), dimethylformamide (“DMF”),chloroform, methylene chloride (or dichloromethane), diethyl ether,methanol, N-methylpyrrolidone (“NMP”), pyridine and the like. Unlessspecified to the contrary, the solvents used in the reactions describedherein are inert organic solvents. Unless specified to the contrary, foreach gram of the limiting reagent, one cc (or mL) of solvent constitutesa volume equivalent.

Isolation and purification of the chemical entities and intermediatesdescribed herein can be effected, if desired, by any suitable separationor purification procedure such as, for example, filtration, extraction,crystallization, column chromatography, thin-layer chromatography orthick-layer chromatography, or a combination of these procedures.Specific illustrations of suitable separation and isolation procedurescan be had by reference to the examples hereinbelow. However, otherequivalent separation or isolation procedures can also be used.

When desired, the (R)- and (S)-isomers of the compounds of the presentinvention, if present, may be resolved by methods known to those skilledin the art, for example by formation of diastereoisomeric salts orcomplexes which may be separated, for example, by crystallization; viaformation of diastereoisomeric derivatives which may be separated, forexample, by crystallization, gas-liquid or liquid chromatography;selective reaction of one enantiomer with an enantiomer-specificreagent, for example enzymatic oxidation or reduction, followed byseparation of the modified and unmodified enantiomers; or gas-liquid orliquid chromatography in a chiral environment, for example on a chiralsupport, such as silica with a bound chiral ligand or in the presence ofa chiral solvent. Alternatively, a specific enantiomer may besynthesized by asymmetric synthesis using optically active reagents,substrates, catalysts or solvents, or by converting one enantiomer tothe other by asymmetric transformation.

The compounds described herein can be optionally contacted with apharmaceutically acceptable acid to form the corresponding acid additionsalts.

Many of the optionally substituted starting compounds and otherreactants are commercially available, e.g., from Aldrich ChemicalCompany (Milwaukee, Wis.) or can be readily prepared by those skilled inthe art using commonly employed synthetic methodology.

The compounds of the invention can generally be synthesized by anappropriate combination of generally well known synthetic methods.Techniques useful in synthesizing these chemical entities are bothreadily apparent and accessible to those of skill in the relevant art,based on the instant disclosure.

The compounds of the invention can be synthesized by an appropriatecombination of known synthetic methods in the art. The discussion belowis offered to illustrate certain of the diverse methods available foruse in making the compounds of the invention and is not intended tolimit the scope of reactions or reaction sequences that can be used inpreparing the compounds of the present invention.

Referring to Scheme 1, Step 1, ketone 101 is converted to thecorresponding alkene using, for example, malononitrile and piperidine inacetic acid. The product, a compound of Formula 102, is isolated.Referring to Scheme 1, Step 2, a compound of Formula 102 is cyclized toa pyridine using, for example, ammonia in methanol. The product, acompound of Formula 103, is isolated. Referring to Scheme 1, Step 3, acompound of Formula 103 is hydrolyzed to the corresponding carboxylicacid. The product, a compound of formula 104, is isolated. Referring toScheme 1, Step 4, a compound of Formula 104 is converted to an amideusing, for example, a standard amide coupling reagent, such as EDCI. Theproduct, a compound of Formula 105, is isolated. Referring to Scheme 1,Step 5, a compound of Formula 105 is converted to the correspondingamide using, for example, chloroacetyl chloride. The product, a compoundof Formula 106, is isolated. Referring to Scheme 1, Step 6, a compoundof Formula 106 is converted to a compound of Formula 107, using, forexample, acetic acid. The product, a compound of Formula 107, isisolated. Referring to Scheme 1, Step 7, a compound of Formula 107undergoes a displacement at the chlorine when reacted with a nucleophileand a base, such as pyrazolopyrimidine 108 and potassium carbonate. Theproduct, a compound of Formula 109, is isolated. Referring to Scheme 1,Step 8, a compound of Formula 109 is coupled with an aryl or heteroarylboronic acid or boronic acid derivative such as, for example, a borolan,using, for example, palladium catalyzed coupling conditions. Theproduct, a compound of Formula 110, is isolated.

Referring to Scheme 2, Step 1, a compound of Formula 108 is converted toa compound of Formula 202, for example, via an alkylation of a compoundof Formula 201. The product, a compound of Formula 202, is isolated.Referring to Scheme 2, Step 2, a compound of Formula 202 is converted toa compound of Formula 203, for example, via saponification. The product,a compound of Formula 203, is isolated. Referring to Scheme 2, Step 3, acompound of Formula 203 is cyclized to a quinazoline of Formula 205, forexample, via sealed tube reaction with a compound of Formula 204 and adehydrating agent, such as PCl₅. The product, a compound of Formula 205,is isolated.

Referring to Scheme 3, a compound of Formula 109 is converted to acompound of Formula 302, for example, via a Sonagashira coupling with acompound of Formula 301. The product, a compound of Formula 302, isisolated.

Referring to Scheme 4, Step 1, a compound of Formula 401 is converted toa compound of Formula 403, for example, via a two step process of Heckcoupling with a compound of Formula 402, followed by acid catalyzedcyclization in methanol. The product, a compound of Formula 403, isisolated. Referring to Scheme 4, Step 2, a compound of Formula 403 isconverted to a compound of Formula 404, for example, via reaction withan appropriately substituted aniline. The product, a compound of Formula404, is isolated. Referring to Scheme 4, Step 3, a compound of Formula404 is converted to a compound of Formula 405, for example, thoughreduction with lithium aluminum hydride. The product, a compound ofFormula 405, is isolated. Referring to Scheme 4, Step 4, a compound ofFormula 405 is converted to a compound of Formula 406, for example, viareaction with thionyl chloride. The product, a compound of Formula 406,is isolated. Referring to Scheme 4, Step 5, a compound of Formula 406 isconverted to a compound of Formula 407, for example, via alkylation witha pyrrazolopyrimidine using a base such as potassium carbonate. Theproduct, a compound of Formula 407, is isolated. Referring to Scheme 4,Step 6, a compound of Formula 407 is converted to a compound of Formula408, for example, via a Suzuki reaction. The product, a compound ofFormula 408, is isolated.

Referring to Scheme 5, Step 1, a compound of Formula 501 is converted toa compound of Formula 502, for example, with a reagent suitable forintroduction of an acid chloride, for example, oxalyl chloride. Theproduct, a compound of Formula 502, is optionally isolated. Referring toScheme 5, Step 2, a compound of Formula 502 is converted to a compoundof Formula 503 for example, reaction with, for example, an aryl amine.The product, a compound of Formula 503, is isolated. Referring to Scheme5, Step 3, a compound of Formula 503 is converted to a compound ofFormula 504, for example, via a Stille coupling using an appropriatevinyl-stannane. The product, a compound of Formula 504, is isolated.Referring to Scheme 5, Step 4, a compound of Formula 504 is converted toa tertiary amide, a compound of Formula 505, via reaction withchloroethyl acetate and sodium hydride base. The compound of Formula 505is isolated. Referring to Scheme 5, Step 5, a compound of Formula 505 isoxidized to an aldehyde, using, for example, osmium tetraoxide andsodium periodinate. The product, a compound of Formula 506, is isolated.Referring to Scheme 5, Step 6, a compound of Formula 506 is converted toa compound of Formula 404, for example, though aldol reaction in ethanolwith a base, such as cesium carbonate. The product, a compound ofFormula 404, is isolated. Referring to Scheme 5, Step 7, a compound ofFormula 404 is reduced to a primary alcohol via reduction with, forexample, lithium aluminum hydride, to produce a compound of Formula 405,which is isolated. Referring to Scheme 5, Step 8, a compound of Formula405 is converted to a compound of Formula 507 via reaction with carbontetrabromide and triphenylphosphine. The compound of Formula 507 isoptionally isolated. This compound can be a central intermediate in thesynthesis of the compounds of the invention.

Referring to Scheme 6, Step 1, a compound of Formula 507, synthesized asdescribed in Reaction Scheme 5 is converted to a compound of Formula 407via coupling with a compound of Formula 108 in the presence of base, forexample, potassium t-butoxide. The compound of Formula 407 is isolated.Referring to Scheme 6, Step 2, a compound of Formula 407 is converted toa compound of Formula 408 via coupling with, for example, an arylboronic acid, in the presence of coupling catalysts and base, forexample, palladium acetate, triphenylphosphine and sodium carbonate, forexample. The compound of Formula 408 is isolated.

Referring to Scheme 7, Step 1, a compound of Formula 701 is reacted witha compound of Formula 702. The product, a compound of Formula 703, isisolated. Referring to Scheme 7, Step 2, a compound of Formula 703 isreacted with an optionally substituted purine such as a compound ofFormula 704. The product, a compound of Formula 705, is isolated.

Referring to Scheme 8, Step 1, a compound of Formula 801 is treated witha reagent such as thionyl chloride to produce a compound of Formula 802,which, is isolated. Referring to Scheme 8, Step 2, a compound of Formula802 and a compound of Formula 803 are combined in the presence of base.The product, a compound of Formula 804, is isolated. Referring to Scheme8, Step 3, a compound of Formula 804 is converted to a compound ofFormula 805, which is isolated.

Referring to Scheme 9, Step 1, a compound of Formula 104 is reacted, forexample, with sodium nitrite and potassium iodide under acidicconditions, to produce a compound of Formula 901, which can be isolated.Referring to Scheme 9, Step 2, the compound of Formula 901 is convertedto its acid chloride by reaction with, for example, oxalyl chloride, toobtain a compound of Formula 902, which can be isolated. Referring toScheme 9, Step 3, the acid chloride of Formula 902 is reacted with anoptionally substituted amino-aryl or amino-hetaryl, compound 903, toyield a compound of Formula 904, which is isolated. Referring to Scheme9, Step 4, the compound of Formula 904 is coupled, for example, with anallyl stannane, to produce a compound of Formula 905, which is isolated.Referring to Scheme 9, Step 5, the compound of Formula 905 is convertedto its epoxide by treatment with, for example, meta-chloroperbenzoicacid, to yield a compound of Formula 906, which can be isolated.Referring to Scheme 9, Step 6, the compound of Formula 906 is cyclizedby treatment, for example, sodium hydride in dimethyl formamide, toobtain a compound of Formula 907, which is isolated. Referring to Scheme9, Step 7, the primary hydroxyl of the compound of Formula 907 isconverted to the bromide by treatment, for example, with carbontetrabromide and triphenylphosphine, to produce a compound of Formula908, which is isolated. Referring to Scheme 9, Step 8, the compound ofFormula 908 is coupled to a pyrazolopyrimidine of Formula 108A bytreatment, for example, with potassium carbonate in dimethylformamide,to yield a compound of Formula 909, which is isolated. Referring toScheme 9, Step 9, the dihydroisoquinolone of Formula 909 is coupled withan optionally substituted aryl or hetaryl boronic acid of Formula 910 toproduce a compound of Formula 911, which is isolated.

Referring to Scheme 10, Step 1, a compound of Formula 1001 is treatedwith a sodium acetate and acetone. The product, a compound of Formula1002, is isolated. Referring to Scheme 10, Step 2, a compound of Formula1002 is cyclized to the corresponding pyrazole, for example, withhydrazine in acetic acid and water. The product, a compound of Formula1003, is isolated. Referring to Scheme 10, Step 3, a compound of Formula1003 is, for example, alkylated using dimethyl sulfate. The product, acompound of Formula 1004, is isolated. Referring to Scheme 10, Step 4, acompound of Formula 1004 is nitrated using, for example, a solution ofnitric acid and sulfuric acid. The product, a compound of Formula 1005,is isolated. Referring to Scheme 10, Step 5, a compound of Formula 1005is saponified using a base, such as sodium hydroxide. The product, acompound of Formula 1006, is isolated. Referring to Scheme 10, Step 6, acompound of Formula 1006 is first converted to an acid chloride usingthionyl chloride, then reacted with an appropriately substituted anilineto create the corresponding amide. The product, a compound of Formula1007, is isolated. Referring to Scheme 10, Step 7, a compound of Formula1007 is reduced to the corresponding amino-pyrazole using hydrogenationconditions with Pd/C as catalyst. The product, a compound of Formula1008, is isolated. Referring to Scheme 10, Step 8, a compound of Formula1008 is cyclized to the corresponding quinazolinone using conditionssuch as chloroacetyl chloride and acetic acid. The product, a compoundof Formula 1009, is isolated. Referring to Scheme 10, Step 9, a compoundof Formula 1009 is coupled with a pyrazolopyrmidine of Formula 108, forexample using conditions such as potassium t-butoxide in DMF at roomtemperature. The product, a compound of Formula 1010, is isolated.Referring to Scheme 10, Step 9, a compound of Formula 1010 is coupledwith an aryl boronic acid of Formula of 910, for example, usingpalladium acetate catalysis, in the presence of triphenyl phosphine andsodium carbonate in DMF to produce a compound of Formula 1011. Theproduct, a compound of Formula 1011, is isolated.

Referring to Scheme 11, Step 1, a compound of Formula 1101 is reactedwith potassium thiocyanide in acetonitrile to produce a compound ofFormula 1102, which can be isolated. Referring to Scheme 11, Step 2, thecompound of Formula 1102 is reacted with a conjugated ester of Formula1103, to produce a compound of Formula 1104, which is isolated.Referring to Scheme 11, Step 3, the compound of Formula 1104 iscyclized, for example, by treating it with bromine in ethanol, to obtaina thiazole of Formula 1105, which is isolated. Referring to Scheme 11,Step 4, the compound of Formula 1105 is deprotected with, for example,potassium carbonate in aqueous dimethylformamide, to yield a compound ofFormula 1106, which is isolated. Referring to Scheme 11, Step 5, theester of the compound of Formula 1106 is saponified with, for example,sodium hydroxide in water, to produce the compound of Formula 1107,which is isolated. Referring to Scheme 11, Step 6, the free acid of thecompound of Formula 1107 is converted to the acid chloride by treatingit with, for example, thionyl chloride, to produce a compound of Formula1108, which can be isolated. Referring to Scheme 11, Step 7, the acidchloride of the compound of Formula 1108 is reacted with an optionallysubstituted amino-aryl or amino-heteroaryl compound of Formula 1109, toobtain a compound of Formula 1110, which is isolated. Referring toScheme 11, Step 8, the primary amine of the compound of Formula 1110 isreacted with a haloacyl chloride, for example, chloroacetyl chloride inpyridine and methylene chloride to produce a compound of Formula 1111,which is isolated. Referring to Scheme 11, Step 9, the compound ofFormula 1111 is cyclized by, for example, heating in a sealed tube inthe presence of phosphoryl chloride, to yield a compound of Formula1112, which is isolated. Referring to Scheme 11, Step 10, thethiazolopyrimidone compound of Formula 1112 is reacted with anpyazolopyrimidine of Formula 1113 in the presence of a base, for examplepotassium t-butoxide in dimethylformamide to produce a compound ofFormula 1114, which is isolated.

Referring to Scheme 12, Step 1, a compound of Formula 709 is alkylatedwith an optionally substituted purine of Formula 1210. The product, acompound of Formula 1211, is isolated.

Referring to Scheme 13, Step 1, a compound of Formula 1301 is firstconverted to an acid chloride using thionyl chloride, then reacted withan appropriately substituted aniline. The product, a compound of Formula1302, is isolated. Referring to Scheme 13, Step 2, a compound of Formula1302 is cyclized to the corresponding thieno-pyrimidinone, for example,with chloroacetyl chloride in acetic acid. The product, a compound ofFormula 1303, is isolated. Referring to Scheme 13, Step 3, a compound ofFormula 1303 is, for example, alkylated using an appropriatelysubstituted pyrazolo-pyrimidine. The product, a compound of Formula1304, is isolated. Referring to Scheme 13, Step 4, a compound of Formula1304 is, for example, arylated using an appropriately substitutedboronic acid. The product, a compound of Formula 1305, is isolated.

Referring to Reaction Scheme 14, Step 1, iodo ester 1401, is reactedwith an alkyne 1402 in the presence of a palladium catalyst, copperiodide and triethylamine (TEA) to couple the alkyne to the aryl core of1401 to produce a compound of Formula 1403. The compound of Formula 1403is isolated. Referring to Reaction Scheme 14, Step 2, a compound ofFormula 1403 is treated with potassium hydroxide base to obtain thecarboxylic acid, a compound of Formula 1404, if the reaction product isacidified, or its salt. The compound of Formula 1404 is isolated.Referring to Reaction Scheme 14, Step 3, a compound of Formula 1404 istreated with bis (acetonitrile)dichloropalladium (II) and TEA to effectintramolecular ring closure to produce a compound of Formula 1405. Thecompound of Formula 1405 is isolated. Referring to Reaction Scheme 14,Step 4, a compound of Formula 1405 is reacted with a primary amine toproduce a compound of Formula 1406. The compound of Formula 1406 isisolated. Referring to Reaction Scheme 14, Step 5, a compound of Formula1406 is treated with hydrochloric acid, removing the protecting group onnitrogen, and to obtain a compound of Formula 1407. The compound ofFormula 1407 can be isolated. Referring to Reaction Scheme 14, Step 6, acompound of Formula 1407 is reacted with a compound of Formula 1408, toproduce a compound of Formula 1409. The compound of Formula 1409 isisolated.

Referring to Reaction Scheme 15, Step 1, iodo ester 1401 is reacted withalkyne 1501 in the presence of palladium coupling catalyst, copperiodide, and TEA, to obtain a compound of Formula 1502. The compound ofFormula 1502 is isolated. Referring to Reaction Scheme 15, Step 2, thecompound of Formula 1502 is treated with potassium hydroxide base toobtain the carboxylate or free acid of a compound of Formula 1503.Referring to Reaction Scheme 15, Step 3, the compound of Formula 1503 istreated with bis (acetonitrile)dichloropalladium (II) and TEA to effectintramolecular ring closure to produce a compound of Formula 1504. Thecompound of Formula 1504 is isolated. Referring to Reaction Scheme 15,Step 4, the compound of Formula 1504 is treated with a primary amine toproduce a compound of Formula 1505. The compound of Formula 1505 isisolated.

Referring to Reaction Scheme 16, Step 1, iodo ester 1401 is reacted withalkyne 1601 in the presence of palladium coupling catalyst, copperiodide, and TEA, to obtain a compound of Formula 1602. The compound ofFormula 1602 is isolated. Referring to Reaction Scheme 16, Step 2, thecompound of Formula 1602 is treated with potassium hydroxide base toobtain the carboxylate or free acid of a compound of Formula 1603.Referring to Reaction Scheme 16, Step 3, the compound of Formula 1603 istreated with bis (acetonitrile)dichoropalladium (II) and TEA to effectintramolecular ring closure to produce a compound of Formula 1604. Thecompound of Formula 1604 is isolated. Referring to Reaction Scheme 16,Step 4, the compound of Formula 1604 is treated with a primary amine toproduce a compound of Formula 1605. The compound of Formula 1605 isisolated. Referring to Reaction Scheme 16, Step 5, the compound ofFormula 1605 is treated with acid to remove the THP protecting group toobtain a compound of Formula 1606. The compound of Formula 1606 isisolated.

Referring to Reaction Scheme 17, Step 1 the compound of Formula 1701 issynthesized by a variety of synthetic routes, including variations ofSchemes 1 or 2 where, for example, a benzyl amine is used in the step ofconverting a compound of Formula 403 to a compound of Formula 404. Thebenzyl protecting group of the amine may be removed by standarddeprotection chemistry to produce a compound of 1701. The compound ofFormula 1701 is converted to a compound of Formula 1702 by alkylation ofthe amide nitrogen with a number of 2-carbon containing synthons whichcan be deprotected, oxidized and reprotected as the respective ketal,the compound of Formula 1702, which can be isolated. Referring toReaction Scheme 17, Step 2-1, the compound of Formula 1702 istransformed by, for example, reductive amination of the ester moiety tointroduce the purinyl moiety of a compound of Formula 1703, oralternatively, is alkylated to so introduce a purinyl moiety and obtaina compound of Formula 1703. Referring to Reaction Scheme 17, Step 3-1,the compound of Formula 1703 is treated with acid to remove the ketalprotecting group to produce a compound of Formula 1704. The compound ofFormula 1704 is isolated. Referring to Reaction Scheme 17, Step 4-1, thecompound of Formula 1704 is reductively aminated with an amine toproduce a compound of Formula 1705. The compound of Formula 1705 isisolated. Referring to Reaction Scheme 17, Step 2-2, the compound ofFormula 1702 is transformed by, steps 7 and 8 of Scheme 5 and step 1 ofScheme 6 to introduce the pyrazolopyrimidine moiety of a compound ofFormula 1706. The compound of Formula 1706 is isolated. Referring toReaction Scheme 17, Step 3-2, the compound of Formula 1706 is treatedwith acid to remove the ketal protecting group to produce a compound ofFormula 1707. The compound of Formula 1707 can be isolated. Referring toReaction Scheme 17, Step 4-2, the compound of Formula 1707 isreductively aminated with an amine to produce a compound of Formula1708. The compound of Formula 1708 is isolated.

Referring to Reaction Scheme 18, Step 1, the compound of Formula 1701 issynthesized as described in Scheme 17 or any other generally knownchemistry. The compound of Formula 1701 is transformed by alkylation ofthe amide nitrogen with a number of 2-carbon containing synthons whichcan be deprotected, and converted to the alkoxy protected species asshown in the compound of Formula 1801, which can be isolated. Referringto Reaction Scheme 18, Step 2, the compound of Formula 1801 is convertedvia chemistry described in Step 2-1 of Scheme 17 to introduce a purinylmoiety, and that resultant compound is transformed by deprotection,activation and amination with an amine to produce a compound of Formula1802, which is isolated.

Referring to Reaction Scheme 18, Step 3, the compound of Formula 1801 isconverted via chemistry described in Step 2-2 of Scheme 17 to introducea pyrazolopyrimidine moiety, and that resultant compound is transformedby deprotection, activation and amination with an amine to produce acompound of Formula 1803, which is isolated.

Referring to Reaction Scheme 19, Step 1, the compound of Formula 1901 istreated with an amine to produce a compound of Formula 1902. Thecompound of Formula 1902 is isolated. Referring to Reaction Scheme 19,Step 2, the compound of Formula 1902 is treated with phosphorusoxychloride to generate a compound of Formula 1903. The compound ofFormula 1903 is isolated. Referring to Reaction Scheme 19, Step 3, thecompound of Formula 1903 is reacted with an amino purine of Formula 1904to obtain a compound of Formula 1905. The compound of Formula 1905 isisolated. Referring to Reaction Scheme 19, Step 4, the compound ofFormula 1905 is treated with hydrochloric acid to remove the protectinggroup at nitrogen on the purine moiety to produce a compound of Formula1906. The compound of 1906 is isolated.

Referring to Reaction Scheme 20, Step 1, the compound of Formula 1401 istreated with vinylogous ester 2001 using, for example a Heck reactionwith subsequent cyclization, to produce a compound of Formula 2002. Thecompound of Formula 2002 is isolated. Referring to Reaction Scheme 20,Step 2, the compound of Formula 2002 is reacted with 4-amino N-Bocpiperidine to produce a compound of Formula 2002. The compound ofFormula 2003 is isolated. The compound of Formula 2003 can be used as anintermediate in the synthesis of the compounds of the invention.

Referring to Reaction Scheme 21, Step 1, the compound of Formula 1401 istreated with an alkynyl alcohol, for example, of Formula 2101, thepresence of copper iodide and palladium on carbon catalyst, to produce acompound of Formula 2102. The compound of Formula 2102 is optionallyisolated and optionally purified. Referring to Reaction Scheme 21, Step1, the compound of Formula 2102 is reacted with 4-amino N-Boc piperidineto produce a compound of Formula 2103. The compound of Formula 2103 isisolated. The compound of Formula 2103 can be used as an intermediate inthe synthesis of the compounds of the invention.

Any of the compounds of Formula I, IV, V, VI, VII, VIII, IX, X, XI, XII,XIII, or XIV can be synthesized using the reaction schemes as disclosedherein or variants of these processes as well known in the art.

The chemical entities can be synthesized by an appropriate combinationof generally well known synthetic methods.

In some embodiments, one or more subject compounds bind specifically toa PI3 kinase or a protein kinase selected from the group consisting ofmTor, DNA-dependent protein kinase DNA-dependent protein kinase (Pubmedprotein accession number (PPAN) AAA79184), Abl tyrosine kinase(CAA52387), Bcr-Abl, hemopoietic cell kinase (PPAN CAI19695), Src (PPANCAA24495), vascular endothelial growth factor receptor 2 (PPANABB82619), vascular endothelial growth factor receptor-2 (PPANABB82619), epidermal growth factor receptor (PPAN AG43241), EPH receptorB4 (PPAN EAL23820), stem cell factor receptor (PPAN AAF22141),Tyrosine-protein kinase receptor TIE-2 (PPAN Q02858), fms-relatedtyrosine kinase 3 (PPAN NP_004110), platelet-derived growth factorreceptor alpha (PPAN NP_990080), RET (PPAN CAA73131), and any otherprotein kinases listed in the appended tables and figures, as well asany functional mutants thereof. In some embodiments, the IC50 of asubject compound for p110α, p110β, p110γ, or p110δ is less than about 1uM, less than about 100 nM, less than about 50 nM, less than about 10nM, less than 1 nM or even less than about 0.5 nM. In some embodiments,the IC50 of a subject compound for mTor is less than about 1 uM, lessthan about 100 nM, less than about 50 nM, less than about 10 nM, lessthan 1 nM or even less than about 0.5 nM. In some other embodiments, oneor more subject compounds exhibit dual binding specificity and arecapable of inhibiting a PI3 kinase (e.g., a class I PI3 kinase) as wellas a protein kinase (e.g., mTor) with an IC50 value less than about 1uM, less than about 100 nM, less than about 50 nM, less than about 10nM, less than 1 nM or even less than about 0.5 nM. One or more subjectcompounds are capable of inhibiting tyrosine kinases including, forexample, DNA-dependent protein kinase DNA-dependent protein kinase(Pubmed protein accession number (PPAN) AAA79184), Abl tyrosine kinase(CAA52387), Bcr-Abl, hemopoietic cell kinase (PPAN CAI19695), Src (PPANCAA24495), vascular endothelial growth factor receptor 2 (PPANABB82619), vascular endothelial growth factor receptor-2 (PPANABB82619), epidermal growth factor receptor (PPAN AG43241), EPH receptorB4 (PPAN EAL23820), stem cell factor receptor (PPAN AAF22141),Tyrosine-protein kinase receptor TIE-2 (PPAN Q02858), fms-relatedtyrosine kinase 3 (PPAN NP_004110), platelet-derived growth factorreceptor alpha (PPAN NP_990080), RET (PPAN CAA73131), and functionalmutants thereof. In some embodiments, the tyrosine kinase is Abl,Bcr-Abl, EGFR, or Flt-3, and any other kinases listed in the Tablesherein.

In some embodiments, the compounds of the present invention exhibits oneor more functional characteristics disclosed herein. For example, one ormore subject compounds bind specifically to a PI3 kinase. In someembodiments, the IC50 of a subject compound for p110α, p110β, p110γ, orp110δ is less than about 1 uM, less than about 100 nM, less than about50 nM, less than about 10 nM, less than about 1 nM, less than about 0.5nM, less than about 100 pM, or less than about 50 pM.

In some embodiments, one or more of the subject compound may selectivelyinhibit one or more members of type I or class I phosphatidylinositol3-kinases (PI3-kinase) with an IC50 value of about 100 nM, 50 nM, 10 nM,5 nM, 100 pM, 10 pM or 1 pM, or less as measured in an in vitro kinaseassay.

In some embodiments, one or more of the subject compound may selectivelyinhibit one or two members of type I or class I phosphatidylinositol3-kinases (PI3-kinase) consisting of PI3-kinase α, PI3-kinase β,PI3-kinase γ, and PI3-kinase δ. In some aspects, some of the subjectcompounds selectively inhibit PI3-kinase δ as compared to all other typeI PI3-kinases. In other aspects, some of the subject compoundsselectively inhibit PI3-kinase δ and PI3-kinase γ as compared to therest of the type I PI3-kinases. In yet other aspects, some of thesubject compounds selectively inhibit PI3-kinase α and PI3-kinase β ascompared to the rest of the type I PI3-kinases. In still yet some otheraspects, some of the subject compounds selectively inhibit PI3-kinase δand PI3-kinase α as compared to the rest of the type I PI3-kinases. Instill yet some other aspects, some of the subject compounds selectivelyinhibit PI3-kinase δ and PI3-kinase β as compared to the rest of thetype I PI3-kinases, or selectively inhibit PI3-kinase δ and PI3-kinase αas compared to the rest of the type I PI3-kinases, or selectivelyinhibit PI3-kinase α and PI3-kinase γ as compared to the rest of thetype I PI3-kinases, or selectively inhibit PI3-kinase γ and PI3-kinase βas compared to the rest of the type I PI3-kinases.

In yet another aspect, an inhibitor that selectively inhibits one ormore members of type I PI3-kinases, or an inhibitor that selectivelyinhibits one or more type I PI3-kinase mediated signaling pathways,alternatively can be understood to refer to a compound that exhibits a50% inhibitory concentration (IC50) with respect to a given type IPI3-kinase, that is at least at least 10-fold, at least 20-fold, atleast 50-fold, at least 100-fold, at least 1000-fold, at least10,100-fold, or lower, than the inhibitor's IC50 with respect to therest of the other type I PI3-kinases.

Pharmaceutical Compositions

The invention provides pharmaceutical compositions comprising one ormore compounds of the present invention.

In some embodiments, the invention provides pharmaceutical compositionsfor treating diseases or conditions related to an undesirable,over-active, harmful or deleterious immune response in a mammal. Suchundesirable immune response can be associated with or result in, e.g.,asthma, emphysema, bronchitis, psoriasis, allergy, anaphylaxsis,auto-immune diseases, rhuematoid arthritis, graft versus host disease,and lupus erythematosus. The pharmaceutical compositions of the presentinvention can be used to treat other respiratory diseases including butnot limited to diseases affecting the lobes of lung, pleural cavity,bronchial tubes, trachea, upper respiratory tract, or the nerves andmuscle for breathing.

In some embodiments, the invention provides pharmaceutical compositionsfor the treatment of disorders such as hyperproliferative disorderincluding but not limited to cancer such as acute myeloid leukemia,thymus, brain, lung, squamous cell, skin, eye, retinoblastoma,intraocular melanoma, oral cavity and oropharyngeal, bladder, gastric,stomach, pancreatic, bladder, breast, cervical, head, neck, renal,kidney, liver, ovarian, prostate, colorectal, esophageal, testicular,gynecological, thyroid, CNS, PNS, AIDS related AIDS-Related (e.g.Lymphoma and Kaposi's Sarcoma) or Viral-Induced cancer. In someembodiments, said pharmaceutical composition is for the treatment of anon-cancerous hyperproliferative disorder such as benign hyperplasia ofthe skin (e. g., psoriasis), restenosis, or prostate (e. g., benignprostatic hypertrophy (BPH)).

The invention also provides compositions for the treatment of liverdiseases (including diabetes), pancreatitis or kidney disease (includingproliferative glomerulonephritis and diabetes-induced renal disease) orpain in a mammal.

The invention further provides a composition for the prevention ofblastocyte implantation in a mammal.

The invention also relates to a composition for treating a diseaserelated to vasculogenesis or angiogenesis in a mammal which can manifestas tumor angiogenesis, chronic inflammatory disease such as rheumatoidarthritis, inflammatory bowel disease, atherosclerosis, skin diseasessuch as psoriasis, eczema, and scleroderma, diabetes, diabeticretinopathy, retinopathy of prematurity, age-related maculardegeneration, hemangioma, glioma, melanoma, Kaposi's sarcoma andovarian, breast, lung, pancreatic, prostate, colon and epidermoidcancer.

The subject pharmaceutical compositions are typically formulated toprovide a therapeutically effective amount of a compound of the presentinvention as the active ingredient, or a pharmaceutically acceptablesalt, ester, prodrug, solvate, hydrate or derivative thereof. Wheredesired, the pharmaceutical compositions contain pharmaceuticallyacceptable salt and/or coordination complex thereof, and one or morepharmaceutically acceptable excipients, carriers, including inert soliddiluents and fillers, diluents, including sterile aqueous solution andvarious organic solvents, permeation enhancers, solubilizers andadjuvants.

The subject pharmaceutical compositions can be administered alone or incombination with one or more other agents, which are also typicallyadministered in the form of pharmaceutical compositions. Where desired,the subject compounds and other agent(s) may be mixed into a preparationor both components may be formulated into separate preparations to usethem in combination separately or at the same time.

In some embodiments, the concentration of one or more of the compoundsprovided in the pharmaceutical compositions of the present invention isless than 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%,16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%,0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%,0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%,0.0004%, 0.0003%, 0.0002%, or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of one or more of the compoundsof the present invention is greater than 90%, 80%, 70%, 60%, 50%, 40%,30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%,17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%,15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%,12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%,10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%,7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%,4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%,1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%,0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%,0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%,0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w/w,w/v, or v/v.

In some embodiments, the concentration of one or more of the compoundsof the present invention is in the range from approximately 0.0001% toapproximately 50%, approximately 0.001% to approximately 40%,approximately 0.01% to approximately 30%, approximately 0.02% toapproximately 29%, approximately 0.03% to approximately 28%,approximately 0.04% to approximately 27%, approximately 0.05% toapproximately 26%, approximately 0.06% to approximately 25%,approximately 0.07% to approximately 24%, approximately 0.08% toapproximately 23%, approximately 0.09% to approximately 22%,approximately 0.1% to approximately 21%, approximately 0.2% toapproximately 20%, approximately 0.3% to approximately 19%,approximately 0.4% to approximately 18%, approximately 0.5% toapproximately 17%, approximately 0.6% to approximately 16%,approximately 0.7% to approximately 15%, approximately 0.8% toapproximately 14%, approximately 0.9% to approximately 12%,approximately 1% to approximately 10% w/w, w/v or v/v. v/v.

In some embodiments, the concentration of one or more of the compoundsof the present invention is in the range from approximately 0.001% toapproximately 10%, approximately 0.01% to approximately 5%,approximately 0.02% to approximately 4.5%, approximately 0.03% toapproximately 4%, approximately 0.04% to approximately 3.5%,approximately 0.05% to approximately 3%, approximately 0.06% toapproximately 2.5%, approximately 0.07% to approximately 2%,approximately 0.08% to approximately 1.5%, approximately 0.09% toapproximately 1%, approximately 0.1% to approximately 0.9% w/w, w/v orv/v.

In some embodiments, the amount of one or more of the compounds of thepresent invention is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g,8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g,3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g,0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g,0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g,0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or 0.0001 g.

In some embodiments, the amount of one or more of the compounds of thepresent invention is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g,0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g,0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g,0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g,0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g,0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g,0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or 10 g.

In some embodiments, the amount of one or more of the compounds of thepresent invention is in the range of 0.0001-10 g, 0.0005-9 g, 0.001-8 g,0.005-7 g, 0.01-6 g, 0.05-5 g, 0.1-4 g, 0.5-4 g, or 1-3 g.

The compounds according to the invention are effective over a widedosage range. For example, in the treatment of adult humans, dosagesfrom 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, andfrom 5 to 40 mg per day are examples of dosages that may be used. Anexemplary dosage is 10 to 30 mg per day. The exact dosage will dependupon the route of administration, the form in which the compound isadministered, the subject to be treated, the body weight of the subjectto be treated, and the preference and experience of the attendingphysician.

Described below are non-limiting exemplary pharmaceutical compositionsand methods for preparing the same.

Pharmaceutical Compositions for Oral Administration

In some embodiments, the invention provides a pharmaceutical compositionfor oral administration containing a compound of the present invention,and a pharmaceutical excipient suitable for oral administration.

In some embodiments, the invention provides a solid pharmaceuticalcomposition for oral administration containing: (i) an effective amountof a compound of the present invention; optionally (ii) an effectiveamount of a second agent; and (iii) a pharmaceutical excipient suitablefor oral administration. In some embodiments, the composition furthercontains: (iv) an effective amount of a third agent.

In some embodiments, the pharmaceutical composition may be a liquidpharmaceutical composition suitable for oral consumption. Pharmaceuticalcompositions of the invention suitable for oral administration can bepresented as discrete dosage forms, such as capsules, cachets, ortablets, or liquids or aerosol sprays each containing a predeterminedamount of an active ingredient as a powder or in granules, a solution,or a suspension in an aqueous or non-aqueous liquid, an oil-in-wateremulsion, or a water-in-oil liquid emulsion. Such dosage forms can beprepared by any of the methods of pharmacy, but all methods include thestep of bringing the active ingredient into association with thecarrier, which constitutes one or more necessary ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelyadmixing the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product intothe desired presentation. For example, a tablet can be prepared bycompression or molding, optionally with one or more accessoryingredients. Compressed tablets can be prepared by compressing in asuitable machine the active ingredient in a free-flowing form such aspowder or granules, optionally mixed with an excipient such as, but notlimited to, a binder, a lubricant, an inert diluent, and/or a surfaceactive or dispersing agent. Molded tablets can be made by molding in asuitable machine a mixture of the powdered compound moistened with aninert liquid diluent.

This invention further encompasses anhydrous pharmaceutical compositionsand dosage forms comprising an active ingredient, since water canfacilitate the degradation of some compounds. For example, water may beadded (e.g., 5%) in the pharmaceutical arts as a means of simulatinglong-term storage in order to determine characteristics such asshelf-life or the stability of formulations over time. Anhydrouspharmaceutical compositions and dosage forms of the invention can beprepared using anhydrous or low moisture containing ingredients and lowmoisture or low humidity conditions. Pharmaceutical compositions anddosage forms of the invention which contain lactose can be madeanhydrous if substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected. An anhydrouspharmaceutical composition may be prepared and stored such that itsanhydrous nature is maintained. Accordingly, anhydrous compositions maybe packaged using materials known to prevent exposure to water such thatthey can be included in suitable formulary kits. Examples of suitablepackaging include, but are not limited to, hermetically sealed foils,plastic or the like, unit dose containers, blister packs, and strippacks.

An active ingredient can be combined in an intimate admixture with apharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. The carrier can take a wide variety of formsdepending on the form of preparation desired for administration. Inpreparing the compositions for an oral dosage form, any of the usualpharmaceutical media can be employed as carriers, such as, for example,water, glycols, oils, alcohols, flavoring agents, preservatives,coloring agents, and the like in the case of oral liquid preparations(such as suspensions, solutions, and elixirs) or aerosols; or carrierssuch as starches, sugars, micro-crystalline cellulose, diluents,granulating agents, lubricants, binders, and disintegrating agents canbe used in the case of oral solid preparations, in some embodimentswithout employing the use of lactose. For example, suitable carriersinclude powders, capsules, and tablets, with the solid oralpreparations. If desired, tablets can be coated by standard aqueous ornonaqueous techniques.

Binders suitable for use in pharmaceutical compositions and dosage formsinclude, but are not limited to, corn starch, potato starch, or otherstarches, gelatin, natural and synthetic gums such as acacia, sodiumalginate, alginic acid, other alginates, powdered tragacanth, guar gum,cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate,carboxymethyl cellulose calcium, sodium carboxymethyl cellulose),polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch,hydroxypropyl methyl cellulose, microcrystalline cellulose, and mixturesthereof.

Examples of suitable fillers for use in the pharmaceutical compositionsand dosage forms disclosed herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.

Disintegrants may be used in the compositions of the invention toprovide tablets that disintegrate when exposed to an aqueousenvironment. Too much of a disintegrant may produce tablets which maydisintegrate in the bottle. Too little may be insufficient fordisintegration to occur and may thus alter the rate and extent ofrelease of the active ingredient(s) from the dosage form. Thus, asufficient amount of disintegrant that is neither too little nor toomuch to detrimentally alter the release of the active ingredient(s) maybe used to form the dosage forms of the compounds disclosed herein. Theamount of disintegrant used may vary based upon the type of formulationand mode of administration, and may be readily discernible to those ofordinary skill in the art. About 0.5 to about 15 weight percent ofdisintegrant, or about 1 to about 5 weight percent of disintegrant, maybe used in the pharmaceutical composition. Disintegrants that can beused to form pharmaceutical compositions and dosage forms of theinvention include, but are not limited to, agar-agar, alginic acid,calcium carbonate, microcrystalline cellulose, croscarmellose sodium,crospovidone, polacrilin potassium, sodium starch glycolate, potato ortapioca starch, other starches, pre-gelatinized starch, other starches,clays, other algins, other celluloses, gums or mixtures thereof.

Lubricants which can be used to form pharmaceutical compositions anddosage forms of the invention include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethylaureate, agar, ormixtures thereof. Additional lubricants include, for example, a syloidsilica gel, a coagulated aerosol of synthetic silica, or mixturesthereof. A lubricant can optionally be added, in an amount of less thanabout 1 weight percent of the pharmaceutical composition.

When aqueous suspensions and/or elixirs are desired for oraladministration, the essential active ingredient therein may be combinedwith various sweetening or flavoring agents, coloring matter or dyesand, if so desired, emulsifying and/or suspending agents, together withsuch diluents as water, ethanol, propylene glycol, glycerin and variouscombinations thereof.

The tablets can be uncoated or coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate canbe employed. Formulations for oral use can also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate orkaolin, or as soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium, for example, peanut oil, liquidparaffin or olive oil.

Surfactant which can be used to form pharmaceutical compositions anddosage forms of the invention include, but are not limited to,hydrophilic surfactants, lipophilic surfactants, and mixtures thereof.That is, a mixture of hydrophilic surfactants may be employed, a mixtureof lipophilic surfactants may be employed, or a mixture of at least onehydrophilic surfactant and at least one lipophilic surfactant may beemployed.

A suitable hydrophilic surfactant may generally have an HLB value of atleast 10, while suitable lipophilic surfactants may generally have anHLB value of or less than about 10. An empirical parameter used tocharacterize the relative hydrophilicity and hydrophobicity of non-ionicamphiphilic compounds is the hydrophilic-lipophilic balance (“HLB”value). Surfactants with lower HLB values are more lipophilic orhydrophobic, and have greater solubility in oils, while surfactants withhigher HLB values are more hydrophilic, and have greater solubility inaqueous solutions. Hydrophilic surfactants are generally considered tobe those compounds having an HLB value greater than about 10, as well asanionic, cationic, or zwitterionic compounds for which the HLB scale isnot generally applicable. Similarly, lipophilic (i.e., hydrophobic)surfactants are compounds having an HLB value equal to or less thanabout 10. However, HLB value of a surfactant is merely a rough guidegenerally used to enable formulation of industrial, pharmaceutical andcosmetic emulsions.

Hydrophilic surfactants may be either ionic or non-ionic. Suitable ionicsurfactants include, but are not limited to, alkylammonium salts;fusidic acid salts; fatty acid derivatives of amino acids,oligopeptides, and polypeptides; glyceride derivatives of amino acids,oligopeptides, and polypeptides; lecithins and hydrogenated lecithins;lysolecithins and hydrogenated lysolecithins; phospholipids andderivatives thereof; lysophospholipids and derivatives thereof;carnitine fatty acid ester salts; salts of alkylsulfates; fatty acidsalts; sodium docusate; acylactylates; mono- and di-acetylated tartaricacid esters of mono- and di-glycerides; succinylated mono- anddi-glycerides; citric acid esters of mono- and di-glycerides; andmixtures thereof.

Within the aforementioned group, ionic surfactants include, by way ofexample: lecithins, lysolecithin, phospholipids, lysophospholipids andderivatives thereof; carnitine fatty acid ester salts; salts ofalkylsulfates; fatty acid salts; sodium docusate; acylactylates; mono-and di-acetylated tartaric acid esters of mono- and di-glycerides;succinylated mono- and di-glycerides; citric acid esters of mono- anddi-glycerides; and mixtures thereof.

Ionic surfactants may be the ionized forms of lecithin, lysolecithin,phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol,phosphatidic acid, phosphatidylserine, lysophosphatidylcholine,lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidicacid, lysophosphatidylserine, PEG-phosphatidylethanolamine,PVP-phosphatidylethanolamine, lactylic esters of fatty acids,stearoyl-2-lactylate, stearoyl lactylate, succinylated monoglycerides,mono/diacetylated tartaric acid esters of mono/diglycerides, citric acidesters of mono/diglycerides, cholylsarcosine, caproate, caprylate,caprate, laurate, myristate, palmitate, oleate, ricinoleate, linoleate,linolenate, stearate, lauryl sulfate, teracecyl sulfate, docusate,lauroyl carnitines, palmitoyl carnitines, myristoyl carnitines, andsalts and mixtures thereof.

Hydrophilic non-ionic surfactants may include, but not limited to,alkylglucosides; alkylmaltosides; alkylthioglucosides; laurylmacrogolglycerides; polyoxyalkylene alkyl ethers such as polyethyleneglycol alkyl ethers; polyoxyalkylene alkylphenols such as polyethyleneglycol alkyl phenols; polyoxyalkylene alkyl phenol fatty acid esterssuch as polyethylene glycol fatty acids monoesters and polyethyleneglycol fatty acids diesters; polyethylene glycol glycerol fatty acidesters; polyglycerol fatty acid esters; polyoxyalkylene sorbitan fattyacid esters such as polyethylene glycol sorbitan fatty acid esters;hydrophilic transesterification products of a polyol with at least onemember of the group consisting of glycerides, vegetable oils,hydrogenated vegetable oils, fatty acids, and sterols; polyoxyethylenesterols, derivatives, and analogues thereof; polyoxyethylated vitaminsand derivatives thereof; polyoxyethylene-polyoxypropylene blockcopolymers; and mixtures thereof; polyethylene glycol sorbitan fattyacid esters and hydrophilic transesterification products of a polyolwith at least one member of the group consisting of triglycerides,vegetable oils, and hydrogenated vegetable oils. The polyol may beglycerol, ethylene glycol, polyethylene glycol, sorbitol, propyleneglycol, pentaerythritol, or a saccharide.

Other hydrophilic-non-ionic surfactants include, without limitation,PEG-10 laurate, PEG-12 laurate, PEG-20 laurate, PEG-32 laurate, PEG-32dilaurate, PEG-12 oleate, PEG-15 oleate, PEG-20 oleate, PEG-20 dioleate,PEG-32 oleate, PEG-200 oleate, PEG-400 oleate, PEG-15 stearate, PEG-32distearate, PEG-40 stearate, PEG-100 stearate, PEG-20 dilaurate, PEG-25glyceryl trioleate, PEG-32 dioleate, PEG-20 glyceryl laurate, PEG-30glyceryl laurate, PEG-20 glyceryl stearate, PEG-20 glyceryl oleate,PEG-30 glyceryl oleate, PEG-30 glyceryl laurate, PEG-40 glyceryllaurate, PEG-40 palm kernel oil, PEG-50 hydrogenated castor oil, PEG-40castor oil, PEG-35 castor oil, PEG-60 castor oil, PEG-40 hydrogenatedcastor oil, PEG-60 hydrogenated castor oil, PEG-60 corn oil, PEG-6caprate/caprylate glycerides, PEG-8 caprate/caprylate glycerides,polyglyceryl-10 laurate, PEG-30 cholesterol, PEG-25 phyto sterol, PEG-30soya sterol, PEG-20 trioleate, PEG-40 sorbitan oleate, PEG-80 sorbitanlaurate, polysorbate 20, polysorbate 80, POE-9 lauryl ether, POE-23lauryl ether, POE-10 oleyl ether, POE-20 oleyl ether, POE-20 stearylether, tocopheryl PEG-100 succinate, PEG-24 cholesterol,polyglyceryl-10oleate, Tween 40, Tween 60, sucrose monostearate, sucrosemonolaurate, sucrose monopalmitate, PEG 10-100 nonyl phenol series, PEG15-100 octyl phenol series, and poloxamers.

Suitable lipophilic surfactants include, by way of example only: fattyalcohols; glycerol fatty acid esters; acetylated glycerol fatty acidesters; lower alcohol fatty acids esters; propylene glycol fatty acidesters; sorbitan fatty acid esters; polyethylene glycol sorbitan fattyacid esters; sterols and sterol derivatives; polyoxyethylated sterolsand sterol derivatives; polyethylene glycol alkyl ethers; sugar esters;sugar ethers; lactic acid derivatives of mono- and di-glycerides;hydrophobic transesterification products of a polyol with at least onemember of the group consisting of glycerides, vegetable oils,hydrogenated vegetable oils, fatty acids and sterols; oil-solublevitamins/vitamin derivatives; and mixtures thereof. Within this group,preferred lipophilic surfactants include glycerol fatty acid esters,propylene glycol fatty acid esters, and mixtures thereof, or arehydrophobic transesterification products of a polyol with at least onemember of the group consisting of vegetable oils, hydrogenated vegetableoils, and triglycerides.

In one embodiment, the composition may include a solubilizer to ensuregood solubilization and/or dissolution of the compound of the presentinvention and to minimize precipitation of the compound of the presentinvention. This can be especially important for compositions fornon-oral use, e.g., compositions for injection. A solubilizer may alsobe added to increase the solubility of the hydrophilic drug and/or othercomponents, such as surfactants, or to maintain the composition as astable or homogeneous solution or dispersion.

Examples of suitable solubilizers include, but are not limited to, thefollowing: alcohols and polyols, such as ethanol, isopropanol, butanol,benzyl alcohol, ethylene glycol, propylene glycol, butanediols andisomers thereof, glycerol, pentaerythritol, sorbitol, mannitol,transcutol, dimethyl isosorbide, polyethylene glycol, polypropyleneglycol, polyvinylalcohol, hydroxypropyl methylcellulose and othercellulose derivatives, cyclodextrins and cyclodextrin derivatives;ethers of polyethylene glycols having an average molecular weight ofabout 200 to about 6000, such as tetrahydrofurfuryl alcohol PEG ether(glycofurol) or methoxy PEG; amides and other nitrogen-containingcompounds such as 2-pyrrolidone, 2-piperidone, .epsilon.-caprolactam,N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone, N-alkylpiperidone,N-alkylcaprolactam, dimethylacetamide and polyvinylpyrrolidone; esterssuch as ethyl propionate, tributylcitrate, acetyl triethylcitrate,acetyl tributyl citrate, triethylcitrate, ethyl oleate, ethyl caprylate,ethyl butyrate, triacetin, propylene glycol monoacetate, propyleneglycol diacetate, ε-caprolactone and isomers thereof, δ-valerolactoneand isomers thereof, β-butyrolactone and isomers thereof; and othersolubilizers known in the art, such as dimethyl acetamide, dimethylisosorbide, N-methyl pyrrolidones, monooctanoin, diethylene glycolmonoethyl ether, and water.

Mixtures of solubilizers may also be used. Examples include, but notlimited to, triacetin, triethylcitrate, ethyl oleate, ethyl caprylate,dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone,polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropylcyclodextrins, ethanol, polyethylene glycol 200-100, glycofurol,transcutol, propylene glycol, and dimethyl isosorbide. Particularlypreferred solubilizers include sorbitol, glycerol, triacetin, ethylalcohol, PEG-400, glycofurol and propylene glycol.

The amount of solubilizer that can be included is not particularlylimited. The amount of a given solubilizer may be limited to abioacceptable amount, which may be readily determined by one of skill inthe art. In some circumstances, it may be advantageous to includeamounts of solubilizers far in excess of bioacceptable amounts, forexample to maximize the concentration of the drug, with excesssolubilizer removed prior to providing the composition to a patientusing conventional techniques, such as distillation or evaporation.Thus, if present, the solubilizer can be in a weight ratio of 10%, 25%,50%, 100%, or up to about 200% by weight, based on the combined weightof the drug, and other excipients. If desired, very small amounts ofsolubilizer may also be used, such as 5%, 2%, 1% or even less.Typically, the solubilizer may be present in an amount of about 1% toabout 100%, more typically about 5% to about 25% by weight.

The composition can further include one or more pharmaceuticallyacceptable additives and excipients. Such additives and excipientsinclude, without limitation, detackifiers, anti-foaming agents,buffering agents, polymers, antioxidants, preservatives, chelatingagents, viscomodulators, tonicifiers, flavorants, colorants, odorants,opacifiers, suspending agents, binders, fillers, plasticizers,lubricants, and mixtures thereof.

In addition, an acid or a base may be incorporated into the compositionto facilitate processing, to enhance stability, or for other reasons.Examples of pharmaceutically acceptable bases include amino acids, aminoacid esters, ammonium hydroxide, potassium hydroxide, sodium hydroxide,sodium hydrogen carbonate, aluminum hydroxide, calcium carbonate,magnesium hydroxide, magnesium aluminum silicate, synthetic aluminumsilicate, synthetic hydrocalcite, magnesium aluminum hydroxide,diisopropylethylamine, ethanolamine, ethylenediamine, triethanolamine,triethylamine, triisopropanolamine, trimethylamine,tris(hydroxymethyl)aminomethane (TRIS) and the like. Also suitable arebases that are salts of a pharmaceutically acceptable acid, such asacetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonicacid, amino acids, ascorbic acid, benzoic acid, boric acid, butyricacid, carbonic acid, citric acid, fatty acids, formic acid, fumaricacid, gluconic acid, hydroquinosulfonic acid, isoascorbic acid, lacticacid, maleic acid, oxalic acid, para-bromophenylsulfonic acid, propionicacid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinicacid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonicacid, uric acid, and the like. Salts of polyprotic acids, such as sodiumphosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphatecan also be used. When the base is a salt, the cation can be anyconvenient and pharmaceutically acceptable cation, such as ammonium,alkali metals, alkaline earth metals, and the like. Example may include,but not limited to, sodium, potassium, lithium, magnesium, calcium andammonium.

Suitable acids are pharmaceutically acceptable organic or inorganicacids. Examples of suitable inorganic acids include hydrochloric acid,hydrobromic acid, hydriodic acid, sulfuric acid, nitric acid, boricacid, phosphoric acid, and the like. Examples of suitable organic acidsinclude acetic acid, acrylic acid, adipic acid, alginic acid,alkanesulfonic acids, amino acids, ascorbic acid, benzoic acid, boricacid, butyric acid, carbonic acid, citric acid, fatty acids, formicacid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbicacid, lactic acid, maleic acid, methanesulfonic acid, oxalic acid,para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid,salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid,thioglycolic acid, toluenesulfonic acid, uric acid and the like.

Pharmaceutical Compositions for Injection.

In some embodiments, the invention provides a pharmaceutical compositionfor injection containing a compound of the present invention and apharmaceutical excipient suitable for injection. Components and amountsof agents in the compositions are as described herein.

The forms in which the novel compositions of the present invention maybe incorporated for administration by injection include aqueous or oilsuspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, orpeanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueoussolution, and similar pharmaceutical vehicles.

Aqueous solutions in saline are also conventionally used for injection.Ethanol, glycerol, propylene glycol, liquid polyethylene glycol, and thelike (and suitable mixtures thereof), cyclodextrin derivatives, andvegetable oils may also be employed. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithin, forthe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like.

Sterile injectable solutions are prepared by incorporating the compoundof the present invention in the required amount in the appropriatesolvent with various other ingredients as enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the various sterilized active ingredients into asterile vehicle which contains the basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions,certain desirable methods of preparation are vacuum-drying andfreeze-drying techniques which yield a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Pharmaceutical Compositions for Topical (e.g., Transdermal) Delivery.

In some embodiments, the invention provides a pharmaceutical compositionfor transdermal delivery containing a compound of the present inventionand a pharmaceutical excipient suitable for transdermal delivery.

Compositions of the present invention can be formulated intopreparations in solid, semi-solid, or liquid forms suitable for local ortopical administration, such as gels, water soluble jellies, creams,lotions, suspensions, foams, powders, slurries, ointments, solutions,oils, pastes, suppositories, sprays, emulsions, saline solutions,dimethylsulfoxide (DMSO)-based solutions. In general, carriers withhigher densities are capable of providing an area with a prolongedexposure to the active ingredients. In contrast, a solution formulationmay provide more immediate exposure of the active ingredient to thechosen area.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients, which are compounds that allow increasedpenetration of, or assist in the delivery of, therapeutic moleculesacross the stratum corneum permeability barrier of the skin. There aremany of these penetration-enhancing molecules known to those trained inthe art of topical formulation. Examples of such carriers and excipientsinclude, but are not limited to, humectants (e.g., urea), glycols (e.g.,propylene glycol), alcohols (e.g., ethanol), fatty acids (e.g., oleicacid), surfactants (e.g., isopropyl myristate and sodium laurylsulfate), pyrrolidones, glycerol monolaurate, sulfoxides, terpenes(e.g., menthol), amines, amides, alkanes, alkanols, water, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin, and polymers such as polyethylene glycols.

Another exemplary formulation for use in the methods of the presentinvention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of a compound of the present invention in controlled amounts,either with or without another agent.

The construction and use of transdermal patches for the delivery ofpharmaceutical agents is well known in the art. See, e.g., U.S. Pat.Nos. 5,023,252, 4,992,445 and 5,001,139. Such patches may be constructedfor continuous, pulsatile, or on demand delivery of pharmaceuticalagents.

Pharmaceutical Compositions for Inhalation.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. Preferably the compositions are administered by the oral or nasalrespiratory route for local or systemic effect. Compositions inpreferably pharmaceutically acceptable solvents may be nebulized by useof inert gases. Nebulized solutions may be inhaled directly from thenebulizing device or the nebulizing device may be attached to a facemask tent, or intermittent positive pressure breathing machine.Solution, suspension, or powder compositions may be administered,preferably orally or nasally, from devices that deliver the formulationin an appropriate manner.

Other Pharmaceutical Compositions.

Pharmaceutical compositions may also be prepared from compositionsdescribed herein and one or more pharmaceutically acceptable excipientssuitable for sublingual, buccal, rectal, intraosseous, intraocular,intranasal, epidural, or intraspinal administration. Preparations forsuch pharmaceutical compositions are well-known in the art. See, e.g.,See, e.g., Anderson, Philip O.; Knoben, James E.; Troutman, William G,eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw-Hill, 2002;Pratt and Taylor, eds., Principles of Drug Action, Third Edition,Churchill Livingston, N.Y., 1990; Katzung, ed., Basic and ClinicalPharmacology, Ninth Edition, McGraw Hill, 20037ybg; Goodman and Gilman,eds., The Pharmacological Basis of Therapeutics, Tenth Edition, McGrawHill, 2001; Remingtons Pharmaceutical Sciences, 20th Ed., LippincottWilliams & Wilkins., 2000; Martindale, The Extra Pharmacopoeia,Thirty-Second Edition (The Pharmaceutical Press, London, 1999); all ofwhich are incorporated by reference herein in their entirety.

Administration of the compounds or pharmaceutical composition of thepresent invention can be effected by any method that enables delivery ofthe compounds to the site of action. These methods include oral routes,intraduodenal routes, parenteral injection (including intravenous,intraarterial, subcutaneous, intramuscular, intravascular,intraperitoneal or infusion), topical (e.g. transdermal application),rectal administration, via local delivery by catheter or stent orthrough inhalation. Compounds can also be administered intraadiposallyor intrathecally.

The amount of the compound administered will be dependent on the mammalbeing treated, the severity of the disorder or condition, the rate ofadministration, the disposition of the compound and the discretion ofthe prescribing physician. However, an effective dosage is in the rangeof about 0.001 to about 100 mg per kg body weight per day, preferablyabout 1 to about 35 mg/kg/day, in single or divided doses. For a 70 kghuman, this would amount to about 0.05 to 7 g/day, preferably about 0.05to about 2.5 g/day. In some instances, dosage levels below the lowerlimit of the aforesaid range may be more than adequate, while in othercases still larger doses may be employed without causing any harmfulside effect, e.g. by dividing such larger doses into several small dosesfor administration throughout the day.

In some embodiments, a compound of the invention is administered in asingle dose. Typically, such administration will be by injection, e.g.,intravenous injection, in order to introduce the agent quickly. However,other routes may be used as appropriate. A single dose of a compound ofthe invention may also be used for treatment of an acute condition.

In some embodiments, a compound of the invention is administered inmultiple doses. Dosing may be about once, twice, three times, fourtimes, five times, six times, or more than six times per day. Dosing maybe about once a month, once every two weeks, once a week, or once everyother day. In another embodiment a compound of the invention and anotheragent are administered together about once per day to about 6 times perday. In another embodiment the administration of a compound of theinvention and an agent continues for less than about 7 days. In yetanother embodiment the administration continues for more than about 6,10, 14, 28 days, two months, six months, or one year. In some cases,continuous dosing is achieved and maintained as long as necessary.

Administration of the agents of the invention may continue as long asnecessary. In some embodiments, an agent of the invention isadministered for more than 1, 2, 3, 4, 5, 6, 7, 14, or 28 days. In someembodiments, an agent of the invention is administered for less than 28,14, 7, 6, 5, 4, 3, 2, or 1 day. In some embodiments, an agent of theinvention is administered chronically on an ongoing basis, e.g., for thetreatment of chronic effects.

An effective amount of a compound of the invention may be administeredin either single or multiple doses by any of the accepted modes ofadministration of agents having similar utilities, including rectal,buccal, intranasal and transdermal routes, by intra-arterial injection,intravenously, intraperitoneally, parenterally, intramuscularly,subcutaneously, orally, topically, or as an inhalant.

The compositions of the invention may also be delivered via animpregnated or coated device such as a stent, for example, or anartery-inserted cylindrical polymer. Such a method of administrationmay, for example, aid in the prevention or amelioration of restenosisfollowing procedures such as balloon angioplasty. Without being bound bytheory, compounds of the invention may slow or inhibit the migration andproliferation of smooth muscle cells in the arterial wall whichcontribute to restenosis. A compound of the invention may beadministered, for example, by local delivery from the struts of a stent,from a stent graft, from grafts, or from the cover or sheath of a stent.In some embodiments, a compound of the invention is admixed with amatrix. Such a matrix may be a polymeric matrix, and may serve to bondthe compound to the stent. Polymeric matrices suitable for such use,include, for example, lactone-based polyesters or copolyesters such aspolylactide, polycaprolactonglycolide, polyorthoesters, polyanhydrides,polyaminoacids, polysaccharides, polyphosphazenes, poly (ether-ester)copolymers (e.g. PEO-PLLA); polydimethylsiloxane,poly(ethylene-vinylacetate), acrylate-based polymers or copolymers (e.g.polyhydroxyethyl methylmethacrylate, polyvinyl pyrrolidinone),fluorinated polymers such as polytetrafluoroethylene and celluloseesters. Suitable matrices may be nondegrading or may degrade with time,releasing the compound or compounds. Compounds of the invention may beapplied to the surface of the stent by various methods such as dip/spincoating, spray coating, dip-coating, and/or brush-coating. The compoundsmay be applied in a solvent and the solvent may be allowed to evaporate,thus forming a layer of compound onto the stent. Alternatively, thecompound may be located in the body of the stent or graft, for examplein microchannels or micropores. When implanted, the compound diffusesout of the body of the stent to contact the arterial wall. Such stentsmay be prepared by dipping a stent manufactured to contain suchmicropores or microchannels into a solution of the compound of theinvention in a suitable solvent, followed by evaporation of the solvent.Excess drug on the surface of the stent may be removed via an additionalbrief solvent wash. In yet other embodiments, compounds of the inventionmay be covalently linked to a stent or graft. A covalent linker may beused which degrades in vivo, leading to the release of the compound ofthe invention. Any bio-labile linkage may be used for such a purpose,such as ester, amide or anhydride linkages. Compounds of the inventionmay additionally be administered intravascularly from a balloon usedduring angioplasty. Extravascular administration of the compounds viathe pericard or via advential application of formulations of theinvention may also be performed to decrease restenosis.

A variety of stent devices which may be used as described are disclosed,for example, in the following references, all of which are herebyincorporated by reference: U.S. Pat. No. 5,451,233; U.S. Pat. No.5,040,548; U.S. Pat. No. 5,061,273; U.S. Pat. No. 5,496,346; U.S. Pat.No. 5,292,331; U.S. Pat. No. 5,674,278; U.S. Pat. No. 3,657,744; U.S.Pat. No. 4,739,762; U.S. Pat. No. 5,195,984; U.S. Pat. No. 5,292,331;U.S. Pat. No. 5,674,278; U.S. Pat. No. 5,879,382; U.S. Pat. No.6,344,053.

The compounds of the invention may be administered in dosages. It isknown in the art that due to intersubject variability in compoundpharmacokinetics, individualization of dosing regimen is necessary foroptimal therapy. Dosing for a compound of the invention may be found byroutine experimentation in light of the instant disclosure.

When a compound of the invention, is administered in a composition thatcomprises one or more agents, and the agent has a shorter half-life thanthe compound of the invention unit dose forms of the agent and thecompound of the invention may be adjusted accordingly.

The subject pharmaceutical composition may, for example, be in a formsuitable for oral administration as a tablet, capsule, pill, powder,sustained release formulations, solution, suspension, for parenteralinjection as a sterile solution, suspension or emulsion, for topicaladministration as an ointment or cream or for rectal administration as asuppository. The pharmaceutical composition may be in unit dosage formssuitable for single administration of precise dosages. Thepharmaceutical composition will include a conventional pharmaceuticalcarrier or excipient and a compound according to the invention as anactive ingredient. In addition, it may include other medicinal orpharmaceutical agents, carriers, adjuvants, etc.

Exemplary parenteral administration forms include solutions orsuspensions of active compound in sterile aqueous solutions, forexample, aqueous propylene glycol or dextrose solutions. Such dosageforms can be suitably buffered, if desired.

The activity of the compounds of the present invention may be determinedby the following procedure, as well as the procedure described in theexamples below. The activity of the kinase is assessed by measuring theincorporation of γ-³³P-phosphate from γ-³³P-ATP onto N-terminal Histagged substrate, which is expressed in E. coli and is purified byconventional methods, in the presence of the kinase. The assay iscarried out in 96-well polypropylene plate. The incubation mixture (100,μL) comprises of 25 mM Hepes, pH 7.4, 10 mM MgCl₂, 5 mMβ-glycerolphosphate, 100 μM Na-orthovanadate, 5 mM DTT, 5 nM kinase, and1 μM substrate. Inhibitors are suspended in DMSO, and all reactions,including controls are performed at a final concentration of 1% DMSO.Reactions are initiated by the addition of 10 μM ATP (with 0.5 μCiγ-³³P-ATP/well) and incubated at ambient temperature for 45 minutes.Equal volume of 25% TCA is added to stop the reaction and precipitatethe proteins. Precipitated proteins are trapped onto glass fiber Bfilterplates, and excess labeled ATP washed off using a Tomtec MACH IIIharvestor. Plates are allowed to air-dry prior to adding 30 μL/well ofPackard Microscint 20, and plates are counted using a Packard TopCount.

The invention also provides kits. The kits include a compound orcompounds of the present invention as described herein, in suitablepackaging, and written material that can include instructions for use,discussion of clinical studies, listing of side effects, and the like.Such kits may also include information, such as scientific literaturereferences, package insert materials, clinical trial results, and/orsummaries of these and the like, which indicate or establish theactivities and/or advantages of the composition, and/or which describedosing, administration, side effects, drug interactions, or otherinformation useful to the health care provider. Such information may bebased on the results of various studies, for example, studies usingexperimental animals involving in vivo models and studies based on humanclinical trials. The kit may further contain another agent. In someembodiments, the compound of the present invention and the agent areprovided as separate compositions in separate containers within the kit.In some embodiments, the compound of the present invention and the agentare provided as a single composition within a container in the kit.Suitable packaging and additional articles for use (e.g., measuring cupfor liquid preparations, foil wrapping to minimize exposure to air, andthe like) are known in the art and may be included in the kit. Kitsdescribed herein can be provided, marketed and/or promoted to healthproviders, including physicians, nurses, pharmacists, formularyofficials, and the like. Kits may also, in some embodiments, be marketeddirectly to the consumer. METHODS

The invention also provides methods of using the compounds orpharmaceutical compositions of the present invention to treat diseaseconditions, including but not limited to diseases associated withmalfunctioning of one or more types of PI3 kinase. A detaileddescription of conditions and disorders mediated by p1106 kinaseactivity is set forth in Sadu et al., WO 01/81346, which is incorporatedherein by reference in its entirety for all purposes.

The treatment methods provided herein comprise administering to thesubject a therapeutically effective amount of a compound of theinvention. In one embodiment, the present invention provides a method oftreating an inflammation disorder, including autoimmune diseases in amammal. The method comprises administering to said mammal atherapeutically effective amount of a compound of the present invention,or a pharmaceutically acceptable salt, ester, prodrug, solvate, hydrateor derivative thereof. Examples of autoimmune diseases includes but isnot limited to acute disseminated encephalomyelitis (ADEM), Addison'sdisease, antiphospholipid antibody syndrome (APS), aplastic anemia,autoimmune hepatitis, coeliac disease, Crohn's disease, Diabetesmellitus (type 1), Goodpasture's syndrome, Graves' disease,Guillain-Barré syndrome (GBS), Hashimoto's disease, lupus erythematosus,multiple sclerosis, myasthenia gravis, opsoclonus myoclonus syndrome(OMS), optic neuritis, Ord's thyroiditis, oemphigus, polyarthritis,primary biliary cirrhosis, psoriasis, rheumatoid arthritis, Reiter'ssyndrome, Takayasu's arteritis, temporal arteritis (also known as “giantcell arteritis”), warm autoimmune hemolytic anemia, Wegener'sgranulomatosis, alopecia universalis, Chagas' disease, chronic fatiguesyndrome, dysautonomia, endometriosis, hidradenitis suppurativa,interstitial cystitis, neuromyotonia, sarcoidosis, scleroderma,ulcerative colitis, vitiligo, and vulvodynia. Other disorders includebone-resorption disorders and thromobsis.

In some embodiments, the method of treating inflammatory or autoimmunediseases comprises administering to a subject (e.g. a mammal) atherapeutically effective amount of one or more compounds of the presentinvention that selectively inhibit PI3K-δ and/or PI3K-γ as compared toall other type I PI3 kinases. Such selective inhibition of PI3K-δ and/orPI3K-γ may be advantageous for treating any of the diseases orconditions described herein. For example, selective inhibition of PI3K-δmay inhibit inflammatory responses associated with inflammatorydiseases, autoimmune disease, or diseases related to an undesirableimmune response including but not limited to asthma, emphysema, allergy,dermatitis, rhuematoid arthritis, psoriasis, lupus erythematosus, orgraft versus host disease. Selective inhibition of PI3K-δ may furtherprovide for a reduction in the inflammatory or undesirable immuneresponse without a concomittant reduction in the ability to reduce abacterial, viral, and/or fungal infection. Selective inhibition of bothPI3K-δ and PI3K-γ may be advantageous for inhibiting the inflammatoryresponse in the subject to a greater degree than that would be providedfor by inhibitors that selectively inhibit PI3K-δ or PI3K-γ alone. Inone aspect, one or more of the subject methods are effective in reducingantigen specific antibody production in vivo by about 2-fold, 3-fold,4-fold, 5-fold, 7.5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 250-fold,500-fold, 750-fold, or about 1000-fold or more. In another aspect, oneor more of the subject methods are effective in reducing antigenspecific IgG3 and/or IgGM production in vivo by about 2-fold, 3-fold,4-fold, 5-fold, 7.5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 250-fold,500-fold, 750-fold, or about 1000-fold or more.

In one aspect, one of more of the subject methods are effective inameliorating symptoms associated with rhuematoid arthritis including butnot limited to a reduction in the swelling of joints, a reduction inserum anti-collagen levels, and/or a reduction in joint pathology suchas bone resorption, cartilage damage, pannus, and/or inflammation. Inanother aspect, the subject methods are effective in reducing ankleinflammation by at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%,60%, or about 75% to 90%. In another aspect, the subject methods areeffective in reducing knee inflammation by at least about 2%, 5%, 10%,15%, 20%, 25%, 30%, 50%, 60%, or about 75% to 90% or more. In stillanother aspect, the subject methods are effective in reducing serumanti-type II collagen levels by at least about 10%, 12%, 15%, 20%, 24%,25%, 30%, 35%, 50%, 60%, 75%, 80%, 86%, 87%, or about 90% or more. Inanother aspect, the subject methods are effective in reducing anklehistopathology scores by about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%,60%, 75%, 80%, 90% or more. In still another aspect, the subject methodsare effective in reducing knee histopathology scores by about 5%, 10%,15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more.

In other embodiments, the present invention provides methods of usingthe compounds or pharmaceutical compositions to treat respiratorydiseases including but not limited to diseases affecting the lobes oflung, pleural cavity, bronchial tubes, trachea, upper respiratory tract,or the nerves and muscle for breathing. For example, methods areprovided to treat obstructive pulmonary disease. Chronic obstructivepulmonary disease (COPD) is an umbrella term for a group of respiratorytract diseases that are characterized by airflow obstruction orlimitation. Conditions included in this umbrella term are: chronicbronchitis, emphysema, and bronchiectasis.

In another embodiment, the compounds described herein are used for thetreatment of asthma. Also, the compounds or pharmaceutical compositionsdescribed herein may be used for the treatment of endotoxemia andsepsis. In one embodiment, the compounds or pharmaceutical compositionsdescribed herein are used to for the treatment of rheumatoid arthritis(RA). In yet another embodiment, the compounds or pharmaceuticalcompositions described herein is used for the treatment of contact oratopic dermatitis. Contact dermatitis includes irritant dermatitis,phototoxic dermatitis, allergic dermatitis, photoallergic dermatitis,contact urticaria, systemic contact-type dermatitis and the like.Irritant dermatitis can occur when too much of a substance is used onthe skin of when the skin is sensitive to certain substance. Atopicdermatitis, sometimes called eczema, is a kind of dermatitis, an atopicskin disease.

The invention also relates to a method of treating a hyperproliferativedisorder in a mammal that comprises administering to said mammal atherapeutically effective amount of a compound of the present invention,or a pharmaceutically acceptable salt, ester, prodrug, solvate, hydrateor derivative thereof. In some embodiments, said method relates to thetreatment of cancer such as acute myeloid leukemia, thymus, brain, lung,squamous cell, skin, eye, retinoblastoma, intraocular melanoma, oralcavity and oropharyngeal, bladder, gastric, stomach, pancreatic,bladder, breast, cervical, head, neck, renal, kidney, liver, ovarian,prostate, colorectal, esophageal, testicular, gynecological, thyroid,CNS, PNS, AIDS-related (e.g. Lymphoma and Kaposi's Sarcoma) orviral-induced cancer. In some embodiments, said method relates to thetreatment of a non-cancerous hyperproliferative disorder such as benignhyperplasia of the skin (e. g., psoriasis), restenosis, or prostate (e.g., benign prostatic hypertrophy (BPH)).

The invention also relates to a method of treating diseases related tovasculogenesis or angiogenesis in a mammal that comprises administeringto said mammal a therapeutically effective amount of a compound of thepresent invention, or a pharmaceutically acceptable salt, ester,prodrug, solvate, hydrate or derivative thereof. In some embodiments,said method is for treating a disease selected from the group consistingof tumor angiogenesis, chronic inflammatory disease such as rheumatoidarthritis, atherosclerosis, inflammatory bowel disease, skin diseasessuch as psoriasis, eczema, and scleroderma, diabetes, diabeticretinopathy, retinopathy of prematurity, age-related maculardegeneration, hemangioma, glioma, melanoma, Kaposi's sarcoma andovarian, breast, lung, pancreatic, prostate, colon and epidermoidcancer.

Patients that can be treated with compounds of the present invention, orpharmaceutically acceptable salt, ester, prodrug, solvate, hydrate orderivative of said compounds, according to the methods of this inventioninclude, for example, patients that have been diagnosed as havingpsoriasis; restenosis; atherosclerosis; BPH; breast cancer such as aductal carcinoma in duct tissue in a mammary gland, medullarycarcinomas, colloid carcinomas, tubular carcinomas, and inflammatorybreast cancer; ovarian cancer, including epithelial ovarian tumors suchas adenocarcinoma in the ovary and an adenocarcinoma that has migratedfrom the ovary into the abdominal cavity; uterine cancer; cervicalcancer such as adenocarcinoma in the cervix epithelial includingsquamous cell carcinoma and adenocarcinomas; prostate cancer, such as aprostate cancer selected from the following: an adenocarcinoma or anadenocarinoma that has migrated to the bone; pancreatic cancer such asepitheliod carcinoma in the pancreatic duct tissue and an adenocarcinomain a pancreatic duct; bladder cancer such as a transitional cellcarcinoma in urinary bladder, urothelial carcinomas (transitional cellcarcinomas), tumors in the urothelial cells that line the bladder,squamous cell carcinomas, adenocarcinomas, and small cell cancers;leukemia such as acute myeloid leukemia (AML), acute lymphocyticleukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairycell leukemia, myelodysplasia, myeloproliferative disorders, acutemyelogenous leukemia (AML), chronic myelogenous leukemia (CML),mastocytosis, chronic lymphocytic leukemia (CLL), multiple myeloma (MM),and myelodysplastic syndrome (MDS); bone cancer; lung cancer such asnon-small cell lung cancer (NSCLC), which is divided into squamous cellcarcinomas, adenocarcinomas, and large cell undifferentiated carcinomas,and small cell lung cancer; skin cancer such as basal cell carcinoma,melanoma, squamous cell carcinoma and actinic keratosis, which is a skincondition that sometimes develops into squamous cell carcinoma; eyeretinoblastoma; cutaneous or intraocular (eye) melanoma; primary livercancer (cancer that begins in the liver); kidney cancer; thyroid cancersuch as papillary, follicular, medullary and anaplastic; AIDS-relatedlymphoma such as diffuse large B-cell lymphoma, B-cell immunoblasticlymphoma and small non-cleaved cell lymphoma; Kaposi's Sarcoma;viral-induced cancers including hepatitis B virus (HBV), hepatitis Cvirus (HCV), and hepatocellular carcinoma; human lymphotropic virus-type1 (HTLV-1) and adult T-cell leukemia/lymphoma; and human papilloma virus(HPV) and cervical cancer; central nervous system cancers (CNS) such asprimary brain tumor, which includes gliomas (astrocytoma, anaplasticastrocytoma, or glioblastoma multiforme), Oligodendroglioma, Ependymoma,Meningioma, Lymphoma, Schwannoma, and Medulloblastoma; peripheralnervous system (PNS) cancers such as acoustic neuromas and malignantperipheral nerve sheath tumor (MPNST) including neurofibromas andschwannomas, malignant fibrous cytoma, malignant fibrous histiocytoma,malignant meningioma, malignant mesothelioma, and malignant mixedMüllerian tumor; oral cavity and oropharyngeal cancer such as,hypopharyngeal cancer, laryngeal cancer, nasopharyngeal cancer, andoropharyngeal cancer; stomach cancer such as lymphomas, gastric stromaltumors, and carcinoid tumors; testicular cancer such as germ cell tumors(GCTs), which include seminomas and nonseminomas, and gonadal stromaltumors, which include Leydig cell tumors and Sertoli cell tumors; thymuscancer such as to thymomas, thymic carcinomas, Hodgkin disease,non-Hodgkin lymphomas carcinoids or carcinoid tumors; rectal cancer; andcolon cancer.

The invention also relates to a method of treating diabetes in a mammalthat comprises administering to said mammal a therapeutically effectiveamount of a compound of the present invention, or a pharmaceuticallyacceptable salt, ester, prodrug, solvate, hydrate or derivative thereof.

In addition, the compounds described herein may be used to treat acne.

In addition, the compounds described herein may be used for thetreatment of arteriosclerosis, including atherosclerosis.Arteriosclerosis is a general term describing any hardening of medium orlarge arteries. Atherosclerosis is a hardening of an artery specificallydue to an atheromatous plaque.

Further the compounds described herein may be used for the treatment ofglomerulonephritis. Glomerulonephritis is a primary or secondaryautoimmune renal disease characterized by inflammation of the glomeruli.It may be asymptomatic, or present with hematuria and/or proteinuria.There are many recognized types, divided in acute, subacute or chronicglomerulonephritis. Causes are infectious (bacterial, viral or parasiticpathogens), autoimmune or paraneoplastic.

Additionally, the compounds described herein may be used for thetreatment of bursitis, lupus, acute disseminated encephalomyelitis(ADEM), addison's disease, antiphospholipid antibody syndrome (APS),aplastic anemia, autoimmune hepatitis, coeliac disease, crohn's disease,diabetes mellitus (type 1), goodpasture's syndrome, graves' disease,guillain-barre syndrome (GBS), hashimoto's disease, inflammatory boweldisease, lupus erythematosus, myasthenia gravis, opsoclonus myoclonussyndrome (OMS), optic neuritis, ord's thyroiditis, ostheoarthritis,uveoretinitis, pemphigus, polyarthritis, primary biliary cirrhosis,reiter's syndrome, takayasu's arteritis, temporal arteritis, warmautoimmune hemolytic anemia, wegener's granulomatosis, alopeciauniversalis, chagas' disease, chronic fatigue syndrome, dysautonomia,endometriosis, hidradenitis suppurativa, interstitial cystitis,neuromyotonia, sarcoidosis, scleroderma, ulcerative colitis, vitiligo,vulvodynia, appendicitis, arteritis, arthritis, blepharitis,bronchiolitis, bronchitis, cervicitis, cholangitis, cholecystitis,chorioamnionitis, colitis, conjunctivitis, cystitis, dacryoadenitis,dermatomyositis, endocarditis, endometritis, enteritis, enterocolitis,epicondylitis, epididymitis, fasciitis, fibrositis, gastritis,gastroenteritis, gingivitis, hepatitis, hidradenitis, ileitis, iritis,laryngitis, mastitis, meningitis, myelitis, myocarditis, myositis,nephritis, omphalitis, oophoritis, orchitis, osteitis, otitis,pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis,pleuritis, phlebitis, pneumonitis, proctitis, prostatitis,pyelonephritis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis,tendonitis, tonsillitis, uveitis, vaginitis, vasculitis, or vulvitis.

The invention also relates to a method of treating a cardiovasculardisease in a mammal that comprises administering to said mammal atherapeutically effective amount of a compound of the present invention,or a pharmaceutically acceptable salt, ester, prodrug, solvate, hydrateor derivative thereof. Examples of cardiovascular conditions include,but are not limited to, atherosclerosis, restenosis, vascular occlusionand carotid obstructive disease.

In another aspect, the present invention provides methods of disruptingthe function of a leukocyte or disrupting a function of an osteoclast.The method includes contacting the leukocyte or the osteoclast with afunction disrupting amount of a compound of the invention.

In another aspect of the present invention, methods are provided fortreating ophthalmic disease by administering one or more of the subjectcompounds or pharmaceutical compositions to the eye of a subject.

Methods are further provided for administering the compounds of thepresent invention via eye drop, intraocular injection, intravitrealinjection, topically, or through the use of a drug eluting device,microcapsule, implant, or microfluidic device. In some cases, thecompounds of the present invention are administered with a carrier orexcipient that increases the intraocular penetrance of the compound suchas an oil and water emulsion with colloid particles having an oily coresurrounded by an interfacial film.

In some cases, the colloid particles include at least one cationic agentand at least one non-ionic sufactant such as a poloxamer, tyloxapol, apolysorbate, a polyoxyethylene castor oil derivative, a sorbitan ester,or a polyoxyl stearate. In some cases, the cationic agent is analkylamine, a tertiary alkyl amine, a quarternary ammonium compound, acationic lipid, an amino alcohol, a biguanidine salt, a cationiccompound or a mixture thereof. In some cases the cationic agent is abiguanidine salt such as chlorhexidine, polyaminopropyl biguanidine,phenformin, alkylbiguanidine, or a mixture thereof. In some cases, thequaternary ammonium compound is a benzalkonium halide, lauralkoniumhalide, cetrimide, hexadecyltrimethylammonium halide,tetradecyltrimethylammonium halide, dodecyltrimethylammonium halide,cetrimonium halide, benzethonium halide, behenalkonium halide,cetalkonium halide, cetethyldimonium halide, cetylpyridinium halide,benzododecinium halide, chlorallyl methenamine halide, myristylalkoniumhalide, stearalkonium halide or a mixture of two or more thereof. Insome cases, cationic agent is a benzalkonium chloride, lauralkoniumchloride, benzododecinium bromide, benzethenium chloride,hexadecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide,dodecyltrimethylammonium bromide or a mixture of two or more thereof. Insome cases, the oil phase is mineral oil and light mineral oil, mediumchain triglycerides (MCT), coconut oil; hydrogenated oils comprisinghydrogenated cottonseed oil, hydrogenated palm oil, hydrogenate castoroil or hydrogenated soybean oil; polyoxyethylene hydrogenated castor oilderivatives comprising poluoxyl-40 hydrogenated castor oil, polyoxyl-60hydrogenated castor oil or polyoxyl-100 hydrogenated castor oil.

The invention further provides methods of modulating kinase activity bycontacting a kinase with an amount of a compound of the inventionsufficient to modulate the activity of the kinase. Modulate can beinhibiting or activating kinase activity. In some embodiments, theinvention provides methods of inhibiting kinase activity by contacting akinase with an amount of a compound of the invention sufficient toinhibit the activity of the kinase. In some embodiments, the inventionprovides methods of inhibiting kinase activity in a solution bycontacting said solution with an amount of a compound of the inventionsufficient to inhibit the activity of the kinase in said solution. Insome embodiments, the invention provides methods of inhibiting kinaseactivity in a cell by contacting said cell with an amount of a compoundof the invention sufficient to inhibit the activity of the kinase insaid cell. In some embodiments, the invention provides methods ofinhibiting kinase activity in a tissue by contacting said tissue with anamount of a compound of the invention sufficient to inhibit the activityof the kinase in said tissue. In some embodiments, the inventionprovides methods of inhibiting kinase activity in an organism bycontacting said organism with an amount of a compound of the inventionsufficient to inhibit the activity of the kinase in said organism. Insome embodiments, the invention provides methods of inhibiting kinaseactivity in an animal by contacting said animal with an amount of acompound of the invention sufficient to inhibit the activity of thekinase in said animal. In some embodiments, the invention providesmethods of inhibiting kinase activity in a mammal by contacting saidmammal with an amount of a compound of the invention sufficient toinhibit the activity of the kinase in said mammal. In some embodiments,the invention provides methods of inhibiting kinase activity in a humanby contacting said human with an amount of a compound of the inventionsufficient to inhibit the activity of the kinase in said human. In someembodiments, the % of kinase activity after contacting a kinase with acompound of the invention is less than 1, 5, 10, 20, 30, 40, 50, 60, 70,80 90, 95, or 99% of the kinase activity in the absence of saidcontacting step.

In some embodiments, the kinase is a lipid kinase or a protein kinase.In some embodiments, the kinase is selected from the group consisting ofPI3 kinase including different isorforms such as PI3 kinase α, PI3kinase β, PI3 kinase γ, PI3 kinase δ; DNA-PK; mTor; Abl, VEGFR, Ephrinreceptor B4 (EphB4); TEK receptor tyrosine kinase (TIE2); FMS-relatedtyrosine kinase 3 (FLT-3); Platelet derived growth factor receptor(PDGFR); RET; ATM; ATR; hSmg-1; Hck; Src; Epidermal growth factorreceptor (EGFR); KIT; Inulsin Receptor (IR) and IGFR.

The invention further provides methods of modulating PI3 kinase activityby contacting a PI3 kinase with an amount of a compound of the inventionsufficient to modulate the activity of the PI3 kinase. Modulate can beinhibiting or activating PI3 kinase activity. In some embodiments, theinvention provides methods of inhibiting PI3 kinase activity bycontacting a PI3 kinase with an amount of a compound of the inventionsufficient to inhibit the activity of the PI3 kinase. In someembodiments, the invention provides methods of inhibiting PI3 kinaseactivity. Such inhibition can take place in solution, in a cellexpressing one or more PI3 kinases, in a tissue comprising a cellexpressing one or more PI3 kinases, or in an organism expressing one ormore PI3 kinases. In some embodiments, the invention provides methods ofinhibiting PI3 kinase activity in an animal (including mammal such ashumans) by contacting said animal with an amount of a compound of theinvention sufficient to inhibit the activity of the PI3 kinase in saidanimal.

Combination Treatment

The present invention also provides methods for combination therapies inwhich an agent known to modulate other pathways, or other components ofthe same pathway, or even overlapping sets of target enzymes are used incombination with a compound of the present invention, or apharmaceutically acceptable salt, ester, prodrug, solvate, hydrate orderivative thereof. In one aspect, such therapy includes but is notlimited to the combination of the subject compound with chemotherapeuticagents, therapeutic antibodies, and radiation treatment, to provide asynergistic or additive therapeutic effect.

In one aspect, the compounds or pharmaceutical compositions of thepresent invention may present synergistic or additive efficacy whenadministered in combination with agents that inhibit IgE production oractivity. Such combination can reduce the undesired effect of high levelof IgE associated with the use of one or more PI3Kδ inhibitors, if sucheffect occurs. This may be particularly useful in treatment ofautoimmune and inflammatory disorders (AIID) such as rheumatoidarthritis. Additionally, the administration of PI3Kδ or PI3Kδ/γinhibitors of the present invention in combination with inhibitors ofmTOR may also exhibit synergy through enhanced inhibition of the PI3Kpathway.

In a separate but related aspect, the present invention provides acombination treatment of a disease associated with PI3Kδ comprisingadministering to a PI3Kδ inhibitor and an agent that inhibits IgEproduction or activity. Other exemplary PI3Kδ inhibitors are applicablefor this combination and they are described, e.g., U.S. Pat. No.6,800,620. Such combination treatment is particularly useful fortreating autoimmune and inflammatory diseases (AIID) including but notlimited to rheumatoid arthritis.

Agents that inhibit IgE production are known in the art and they includebut are not limited to one or more of TEI-9874,2-(4-(6-cyclohexyloxy-2-naphtyloxy)phenylacetamide)benzoic acid,rapamycin, rapamycin analogs (i.e. rapalogs), TORC1 inhibitors, TORC2inhibitors, and any other compounds that inhibit mTORC1 and mTORC2.Agents that inhibit IgE activity include, for example, anti-IgEantibodies such as for example Omalizumab and TNX-901.

For treatment of autoimmune diseases, the subject compounds orpharmaceutical compositions can be used in combination with commonlyprescribed drugs including but not limited to Enbrel®, Remicade®,Humira®, Avonex®, and Rebif®. For treatment of respiratory diseases, thesubject compounds or pharmaceutical compositions can be administered incombination with commonly prescribed drugs including but not limited toXolair®, Advair®, Singulair®, and Spiriva®.

The compounds of the invention may be formulated or administered inconjunction with other agents that act to relieve the symptoms ofinflammatory conditions such as encephalomyelitis, asthma, and the otherdiseases described herein. These agents include non-steroidalanti-inflammatory drugs (NSAIDs), e.g. acetylsalicylic acid; ibuprofen;naproxen; indomethacin; nabumetone; tolmetin; etc. Corticosteroids areused to reduce inflammation and suppress activity of the immune system.The most commonly prescribed drug of this type is Prednisone.Chloroquine (Aralen) or hydroxychloroquine (Plaquenil) may also be veryuseful in some individuals with lupus. They are most often prescribedfor skin and joint symptoms of lupus. Azathioprine (Imuran) andcyclophosphamide (Cytoxan) suppress inflammation and tend to suppressthe immune system. Other agents, e.g. methotrexate and cyclosporin areused to control the symptoms of lupus. Anticoagulants are employed toprevent blood from clotting rapidly. They range from aspirin at very lowdose which prevents platelets from sticking, to heparin/coumadin.

In another one aspect, this invention also relates to a pharmaceuticalcomposition for inhibiting abnormal cell growth in a mammal whichcomprises an amount of a compound of the present invention, or apharmaceutically acceptable salt, ester, prodrug, solvate, hydrate orderivative thereof, in combination with an amount of an anti-canceragent (e.g. a chemotherapeutic agent). Many chemotherapeutics arepresently known in the art and can be used in combination with thecompounds of the invention.

In some embodiments, the chemotherapeutic is selected from the groupconsisting of mitotic inhibitors, alkylating agents, anti-metabolites,intercalating antibiotics, growth factor inhibitors, cell cycleinhibitors, enzymes, topoisomerase inhibitors, biological responsemodifiers, anti-hormones, angiogenesis inhibitors, and anti-androgens.Non-limiting examples are chemotherapeutic agents, cytotoxic agents, andnon-peptide small molecules such as Gleevec (Imatinib Mesylate), Velcade(bortezomib), Casodex (bicalutamide), Iressa (gefitinib), and Adriamycinas well as a host of chemotherapeutic agents. Non-limiting examples ofchemotherapeutic agents include alkylating agents such as thiotepa andcyclosphosphamide (CYTOXAN™); alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, Casodex™, chromomycins, dactinomycin, daunorubicin,detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin,esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid,nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfomithine; elliptinium acetate; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone;mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinicacid; 2-ethylhydrazide; procarbazine; PSK.R™; razoxane; sizofiran;spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethyla-mine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g.paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddocetaxel (TAXOTERE™, Rhone-Poulenc Rorer, Antony, France); retinoicacid; esperamicins; capecitabine; and pharmaceutically acceptable salts,acids or derivatives of any of the above. Also included as suitablechemotherapeutic cell conditioners are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogensincluding for example tamoxifen (Nolvadex™), raloxifene, aromataseinhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,LY 117018, onapristone, and toremifene (Fareston); and anti-androgenssuch as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; camptothecin-11 (CPT-11);topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO). Wheredesired, the compounds or pharmaceutical composition of the presentinvention can be used in combination with commonly prescribedanti-cancer drugs such as Herceptin®, Avastin®, Erbitux®, Rituxan®,Taxol®, Arimidex®, Taxotere®, and Velcade®.

This invention further relates to a method for using the compounds orpharmaceutical composition in combination with radiation therapy ininhibiting abnormal cell growth or treating the hyperproliferativedisorder in the mammal. Techniques for administering radiation therapyare known in the art, and these techniques can be used in thecombination therapy described herein. The administration of the compoundof the invention in this combination therapy can be determined asdescribed herein.

Radiation therapy can be administered through one of several methods, ora combination of methods, including without limitation external-beamtherapy, internal radiation therapy, implant radiation, stereotacticradiosurgery, systemic radiation therapy, radiotherapy and permanent ortemporary interstitial brachytherapy. The term “brachytherapy,” as usedherein, refers to radiation therapy delivered by a spatially confinedradioactive material inserted into the body at or near a tumor or otherproliferative tissue disease site. The term is intended withoutlimitation to include exposure to radioactive isotopes (e.g. At-211,1-131, 1-125, Y-90, Re-186, Re-188, Sm-153, Bi-212, P-32, andradioactive isotopes of Lu). Suitable radiation sources for use as acell conditioner of the present invention include both solids andliquids. By way of non-limiting example, the radiation source can be aradionuclide, such as 1-125, 1-131, Yb-169, Ir-192 as a solid source,1-125 as a solid source, or other radionuclides that emit photons, betaparticles, gamma radiation, or other therapeutic rays. The radioactivematerial can also be a fluid made from any solution of radionuclide(s),e.g., a solution of I-125 or I-131, or a radioactive fluid can beproduced using a slurry of a suitable fluid containing small particlesof solid radionuclides, such as Au-198, Y-90. Moreover, theradionuclide(s) can be embodied in a gel or radioactive micro spheres.

Without being limited by any theory, the compounds of the presentinvention can render abnormal cells more sensitive to treatment withradiation for purposes of killing and/or inhibiting the growth of suchcells. Accordingly, this invention further relates to a method forsensitizing abnormal cells in a mammal to treatment with radiation whichcomprises administering to the mammal an amount of a compound of thepresent invention or pharmaceutically acceptable salt, ester, prodrug,solvate, hydrate or derivative thereof, which amount is effective issensitizing abnormal cells to treatment with radiation. The amount ofthe compound, salt, or solvate in this method can be determinedaccording to the means for ascertaining effective amounts of suchcompounds described herein.

The compounds or pharmaceutical compositions of the present inventioncan be used in combination with an amount of one or more substancesselected from anti-angiogenesis agents, signal transduction inhibitors,and antiproliferative agents.

Anti-angiogenesis agents, such as MMP-2 (matrix-metalloprotienase 2)inhibitors, MMP-9 (matrix-metalloprotienase 9) inhibitors, and COX-11(cyclooxygenase 11) inhibitors, can be used in conjunction with acompound of the present invention and pharmaceutical compositionsdescribed herein. Examples of useful COX-II inhibitors include CELEBREX™(alecoxib), valdecoxib, and rofecoxib. Examples of useful matrixmetalloproteinase inhibitors are described in WO 96/33172 (publishedOct. 24, 1996), WO 96/27583 (published Mar. 7, 1996), European PatentApplication No. 97304971.1 (filed Jul. 8, 1997), European PatentApplication No. 99308617.2 (filed Oct. 29, 1999), WO 98/07697 (publishedFeb. 26, 1998), WO 98/03516 (published Jan. 29, 1998), WO 98/34918(published Aug. 13, 1998), WO 98/34915 (published Aug. 13, 1998), WO98/33768 (published Aug. 6, 1998), WO 98/30566 (published Jul. 16,1998), European Patent Publication 606,046 (published Jul. 13, 1994),European Patent Publication 931, 788 (published Jul. 28, 1999), WO90/05719 (published May 31, 1990), WO 99/52910 (published Oct. 21,1999), WO 99/52889 (published Oct. 21, 1999), WO 99/29667 (publishedJun. 17, 1999), PCT International Application No. PCT/IB98/01113 (filedJul. 21, 1998), European Patent Application No. 99302232.1 (filed Mar.25, 1999), Great Britain Patent Application No. 9912961.1 (filed Jun. 3,1999), U.S. Provisional Application No. 60/148,464 (filed Aug. 12,1999), U.S. Pat. No. 5,863,949 (issued Jan. 26, 1999), U.S. Pat. No.5,861,510 (issued Jan. 19, 1999), and European Patent Publication780,386 (published Jun. 25, 1997), all of which are incorporated hereinin their entireties by reference. Preferred MMP-2 and MMP-9 inhibitorsare those that have little or no activity inhibiting MMP-1. Morepreferred, are those that selectively inhibit MMP-2 and/or AMP-9relative to the other matrix-metalloproteinases (i. e., MAP-1, MMP-3,MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13).Some specific examples of MMP inhibitors useful in the present inventionare AG-3340, RO 32-3555, and RS 13-0830.

The invention also relates to a method of and to a pharmaceuticalcomposition of treating a cardiovascular disease in a mammal whichcomprises an amount of a compound of the present invention, or apharmaceutically acceptable salt, ester, prodrug, solvate, hydrate orderivative thereof, or an isotopically-labeled derivative thereof, andan amount of one or more therapeutic agents use for the treatment ofcardiovascular diseases.

Examples for use in cardiovascular disease applications areanti-thrombotic agents, e.g., prostacyclin and salicylates, thrombolyticagents, e.g., streptokinase, urokinase, tissue plasminogen activator(TPA) and anisoylated plasminogen-streptokinase activator complex(APSAC), anti-platelets agents, e.g., acetyl-salicylic acid (ASA) andclopidrogel, vasodilating agents, e.g., nitrates, calcium channelblocking drugs, anti-proliferative agents, e.g., colchicine andalkylating agents, intercalating agents, growth modulating factors suchas interleukins, transformation growth factor-beta and congeners ofplatelet derived growth factor, monoclonal antibodies directed againstgrowth factors, anti-inflammatory agents, both steroidal andnon-steroidal, and other agents that can modulate vessel tone, function,arteriosclerosis, and the healing response to vessel or organ injurypost intervention. Antibiotics can also be included in combinations orcoatings comprised by the invention. Moreover, a coating can be used toeffect therapeutic delivery focally within the vessel wall. Byincorporation of the active agent in a swellable polymer, the activeagent will be released upon swelling of the polymer.

The compounds describe herein may be formulated or administered inconjunction with liquid or solid tissue barriers also known aslubricants. Examples of tissue barriers include, but are not limited to,polysaccharides, polyglycans, seprafilm, interceed and hyaluronic acid.

Medicaments which may be administered in conjunction with the compoundsdescribed herein include any suitable drugs usefully delivered byinhalation for example, analgesics, e.g. codeine, dihydromorphine,ergotamine, fentanyl or morphine; anginal preparations, e.g. diltiazem;antiallergics, e.g. cromoglycate, ketotifen or nedocromil;anti-infectives, e.g. cephalosporins, penicillins, streptomycin,sulphonamides, tetracyclines or pentamidine; antihistamines, e.g.methapyrilene; anti-inflammatories, e.g. beclomethasone, flunisolide,budesonide, tipredane, triamcinolone acetonide or fluticasone;antitussives, e.g. noscapine; bronchodilators, e.g. ephedrine,adrenaline, fenoterol, formoterol, isoprenaline, metaproterenol,phenylephrine, phenylpropanolamine, pirbuterol, reproterol, rimiterol,salbutamol, salmeterol, terbutalin, isoetharine, tulobuterol,orciprenaline or(−)-4-amino-3,5-dichloro-α-[[[6-[2-(2-pyridinyl)ethoxy]hexyl]-amino]methyl]benzenemethanol;diuretics, e.g. amiloride; anticholinergics e.g. ipratropium, atropineor oxitropium; hormones, e.g. cortisone, hydrocortisone or prednisolone;xanthines e.g. aminophylline, choline theophyllinate, lysinetheophyllinate or theophylline; and therapeutic proteins and peptides,e.g. insulin or glucagon. It will be clear to a person skilled in theart that, where appropriate, the medicaments may be used in the form ofsalts (e.g. as alkali metal or amine salts or as acid addition salts) oras esters (e.g. lower alkyl esters) or as solvates (e.g. hydrates) tooptimize the activity and/or stability of the medicament.

Other exemplary therapeutic agents useful for a combination therapyinclude but are not limited to agents as described above, radiationtherapy, hormone antagonists, hormones and their releasing factors,thyroid and antithyroid drugs, estrogens and progestins, androgens,adrenocorticotropic hormone; adrenocortical steroids and their syntheticanalogs; inhibitors of the synthesis and actions of adrenocorticalhormones, insulin, oral hypoglycemic agents, and the pharmacology of theendocrine pancreas, agents affecting calcification and bone turnover:calcium, phosphate, parathyroid hormone, vitamin D, calcitonin, vitaminssuch as water-soluble vitamins, vitamin B complex, ascorbic acid,fat-soluble vitamins, vitamins A, K, and E, growth factors, cytokines,chemokines, muscarinic receptor agonists and antagonists;anticholinesterase agents; agents acting at the neuromuscular junctionand/or autonomic ganglia; catecholamines, sympathomimetic drugs, andadrenergic receptor agonists or antagonists; and 5-hydroxytryptamine(5-HT, serotonin) receptor agonists and antagonists.

Therapeutic agents can also include agents for pain and inflammationsuch as histamine and histamine antagonists, bradykinin and bradykininantagonists, 5-hydroxytryptamine (serotonin), lipid substances that aregenerated by biotransformation of the products of the selectivehydrolysis of membrane phospholipids, eicosanoids, prostaglandins,thromboxanes, leukotrienes, aspirin, nonsteroidal anti-inflammatoryagents, analgesic-antipyretic agents, agents that inhibit the synthesisof prostaglandins and thromboxanes, selective inhibitors of theinducible cyclooxygenase, selective inhibitors of the induciblecyclooxygenase-2, autacoids, paracrine hormones, somatostatin, gastrin,cytokines that mediate interactions involved in humoral and cellularimmune responses, lipid-derived autacoids, eicosanoids, β-adrenergicagonists, ipratropium, glucocorticoids, methylxanthines, sodium channelblockers, opioid receptor agonists, calcium channel blockers, membranestabilizers and leukotriene inhibitors.

Additional therapeutic agents contemplated herein include diuretics,vasopressin, agents affecting the renal conservation of water, rennin,angiotensin, agents useful in the treatment of myocardial ischemia,anti-hypertensive agents, angiotensin converting enzyme inhibitors,β-adrenergic receptor antagonists, agents for the treatment ofhypercholesterolemia, and agents for the treatment of dyslipidemia.

Other therapeutic agents contemplated include drugs used for control ofgastric acidity, agents for the treatment of peptic ulcers, agents forthe treatment of gastroesophageal reflux disease, prokinetic agents,antiemetics, agents used in irritable bowel syndrome, agents used fordiarrhea, agents used for constipation, agents used for inflammatorybowel disease, agents used for biliary disease, agents used forpancreatic disease. Therapeutic agents used to treat protozoaninfections, drugs used to treat Malaria, Amebiasis, Giardiasis,Trichomoniasis, Trypanosomiasis, and/or Leishmaniasis, and/or drugs usedin the chemotherapy of helminthiasis. Other therapeutic agents includeantimicrobial agents, sulfonamides, trimethoprim-sulfamethoxazolequinolones, and agents for urinary tract infections, penicillins,cephalosporins, and other, β-Lactam antibiotics, an agent comprising anaminoglycoside, protein synthesis inhibitors, drugs used in thechemotherapy of tuberculosis, mycobacterium avium complex disease, andleprosy, antifungal agents, antiviral agents including nonretroviralagents and antiretroviral agents.

Examples of therapeutic antibodies that can be combined with a subjectcompound include but are not limited to anti-receptor tyrosine kinaseantibodies (cetuximab, panitumumab, trastuzumab), anti CD20 antibodies(rituximab, tositumomab), and other antibodies such as alemtuzumab,bevacizumab, and gemtuzumab.

Moreover, therapeutic agents used for immunomodulation, such asimmunomodulators, immunosuppressive agents, tolerogens, andimmunostimulants are contemplated by the methods herein. In addition,therapeutic agents acting on the blood and the blood-forming organs,hematopoietic agents, growth factors, minerals, and vitamins,anticoagulant, thrombolytic, and antiplatelet drugs.

Further therapeutic agents that can be combined with a subject compoundmay be found in Goodman and Gilman's “The Pharmacological Basis ofTherapeutics” Tenth Edition edited by Hardman, Limbird and Gilman or thePhysician's Desk Reference, both of which are incorporated herein byreference in their entirety.

The compounds described herein can be used in combination with theagents disclosed herein or other suitable agents, depending on thecondition being treated. Hence, in some embodiments the compounds of theinvention will be co-administer with other agents as described above.When used in combination therapy, the compounds described herein may beadministered with the second agent simultaneously or separately. Thisadministration in combination can include simultaneous administration ofthe two agents in the same dosage form, simultaneous administration inseparate dosage forms, and separate administration. That is, a compounddescribed herein and any of the agents described above can be formulatedtogether in the same dosage form and administered simultaneously.Alternatively, a compound of the present invention and any of the agentsdescribed above can be simultaneously administered, wherein both theagents are present in separate formulations. In another alternative, acompound of the present invention can be administered just followed byand any of the agents described above, or vice versa. In the separateadministration protocol, a compound of the present invention and any ofthe agents described above may be administered a few minutes apart, or afew hours apart, or a few days apart.

The examples and preparations provided below further illustrate andexemplify the compounds of the present invention and methods ofpreparing such compounds. It is to be understood that the scope of thepresent invention is not limited in any way by the scope of thefollowing examples and preparations. In the following examples moleculeswith a single chiral center, unless otherwise noted, exist as a racemicmixture. Those molecules with two or more chiral centers, unlessotherwise noted, exist as a racemic mixture of diastereomers. Singleenantiomers/diastereomers may be obtained by methods known to thoseskilled in the art.

EXAMPLES Example 1: Synthesis of2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methyl-3-o-tolylpyrido[2,3-d]pyrimidin-4(3H)-one(Compound 1406)

To a stirred solution of 4,4-dimethoxy-2-butanone (101)(61 g, 85%, 0.393mol), acetic acid (2.2 mL, 0.038 mmol) and piperidine (3.8 mL, 0.038mol) in toluene (150 mL), malononitrile (25 g, 0.394 mmol) was added inportions over 20 min. The reaction mixture was stirred overnight at roomtemperature. The resulting dark red solution was washed with H₂O (50mL), dried over MgSO₄ and concentrated in vacuo to afford the desiredproduct 2-(4,4-dimethoxybutan-2-ylidene) malononitrile (102) (70 g,99%), which was used directly in the next step.

Ammonia gas was bubbled through a solution of 102 (32 g, 0.178 mmol) inMeOH (500 mL) for 3 h, the resulting deep-red solution was stirredovernight at room temperature. The mixture was concentrated and theresidue was partitioned between HCl solution (2N, 600 mL) and EtOAc (600mL). The aqueous layer was separated and basified with ice-coldconcentrated NaHCO₃ (600 mL) solution. The solid was precipitated out ofthe solution and collected by filtration to afford the desired product2-amino-4-methylnicotinonitrile (103) (3.0 g, 33%).

Compound 103 (5.32 g, 40 mmol) was suspended in a solution of potassiumhydroxide (26.88 g, 480 mmol) in water (26.85 mL) and iso-propanol (9.6mL). The reaction mixture was heated to reflux for 50 h, then cooled toroom temperature and diluted with ice-water (100 mL) and neutralizedwith concentrated HCl solution until PH=6-7. The mixture wasconcentrated in vacuo and the resulting residue was purified by flashchromatography eluting with ethanol to afford the desired product2-amino-4-methylnicotinic acid (104) (3.284 g, 54.7%).

To a stirred solution of 104 (3.2 g, 21.2 mmol) in DMF (40 mL) and DCM(80 mL) was added EDCI (8.12 g, 42.4 mmol), HOBt (2.86 g, 21.4 mmol) ando-Toluidine (4.53 mL, 42.4 mmol). The reaction mixture was stirredovernight at room temperature and then poured into water (120 mL). Theaqueous phase was extracted with DCM (60 mL×2). The combined organicphases were washed with brine, dried over MgSO₄, filtered andconcentrated. The solid was precipitated out of the solution. The solidwas collected by filtration and dried to afford the desired product2-amino-4-methyl-N-o-tolylnicotinamide (1401) (3.6 g, 70.4%).

A suspension of 1401 (1.2 g, 4.96 mmol) in dry THF (60 mL) was addedbutyllithium (2.5M, 2.38 mL, 5.96 mmol) dropwise under Argon at −40° C.and stirred at this temperature for 1 h. Then the reaction mixture wascooled to −78° C. and chloroacetyl chloride (0.432 mL, 5.4 mmol) wasadded. After stirring for 2 h at −78° C., the reaction mixture waspoured into ice-water (100 mL). After most of THF was removed in vacuo,the solid was precipitated out of the solution. The solid was collectedby filtration and washed with ether to afford the desired product2-(2-chloroacetamido)-4-methyl-N-o-tolylnicotinamide (1402) (890 mg,56.4%).

A mixture of 1402 (320 mg, 1 mmol) and phosphorus oxychloride (20 mL,214 mmol) was heated at 115° C. overnight in a sealed-tube. The reactionmixture was cooled to room temperature and concentrated in vacuo. Theresidue was poured into ice-water and neutralized with saturated NaHCO₃solution until PH 8-9, the resulting precipitate solid was collected byfiltration and washed with ether to afford the desired product2-(chloromethyl)-5-methyl-3-o-tolylpyrido[2,3-d]pyrimidin-4(3H)-one(1403) (200 mg, 66.8%).

To a solution of 3-iodo-4-amine-1H-pyrazolo[3,4-d]pyrimidin (108A)(261mg, 1.2 mmol) in dry DMF (9 mL) under nitrogen, potassiumtert-butoxide(123 mg, 1.1 mmol) was added at 0° C. The resulting mixturewas stirred at this temperature for 45 min. A solution of 1403 (300 mg,1 mmol) in dry DMF (5 mL) was added. The reaction mixture was stirredfor 1 h at 0° C. and then an additional 1 h at room temperature. Themixture was concentrated in vacuo and the resulting residue was purifiedby flash chromatography to afford the desired product2-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methyl-3-o-tolylpyrido[2,3-d]pyrimidin-4(3H)-one(1404) (450 mg, 83.3%).

To a solution of 1404 (36 mg, 0.069 mmol) and 3-hydroxyphenylboronicacid (1405)(12 mg, 0.083 mmol) in DMF (2 mL), EtOH (1 mL) and water (1mL) was added Pd(PPh₃)₄(7 mg, 0.006 mmol) and Na₂CO₃ solution (1M, 0.5mL, 0.5 mmol) under Argon. The resulting mixture was degassed andback-filled with argon three times and then heated overnight at 80° C.The reaction mixture was allowed to cool to room temperature,concentrated. The residue was diluted with water (20 mL), neutralizedwith HCl solution (1M) until pH 6-7 and extracted with DCM (10 mL×3).The combined organic layers were washed with brine, dried over Na₂SO₄and concentrated. The residue was purified by flash chromatography toafford the desired product2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methyl-3-o-tolylpyrido[2,3-d]pyrimidin-4(3H)-one(1406) (8 mg, 23.7%).

Example 2: Synthesis of5-((4-amino-3-(3-fluoro-4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1,3-dimethyl-6-o-tolyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one(Compound 1511)

Sodium (5.2 g, 0.226 mol) was dissolved in anhydrous ethanol (120 mL). Amixture of diethyl oxalate (1501)(31.8 mL, 0.235 mol) and acetone (16.0mL, 0.218 mol) was added to the above solution keeping the temperatureunder 10° C. The reaction mixture was stirred overnight at RT. Theresulting precipitate was collected by filtration, washed withpetroether and dried to afford the desired product 1502 as a yellowsolid. (30.4 g, 77.5%).

Hydrazine hydrate (9.7 mL, 85%, 0.200 mol) was added dropwise to aceticacid (34 mL). To this solution, compound 1502 (30.4 g, 0.169 mol) wasadded in portions at 25° C. The resulting mixture was stirred for 2 h atroom temperature, then basified with saturated NaHCO₃ solution until pH8 and extracted with DCM (200 mL×3). The combined organic phases werewashed with brine, dried and concentrated to afford the desired product,compound 1503, as a yellow solid (22 g, 84.6%).

Dimethyl sulfate (3.2 mL, 33.8 mmol) was added dropwise to a solution ofcompound 1503 (4.2 g, 29.9 mmol) in toluene (20 mL). The reactionmixture was stirred for 4 h at 80° C., then allowed to cool to roomtemperature and concentrated. A 40% NaOH solution (15 mL) was added tothe residue. The resulting mixture was stirred for 1 h at 80° C., thencooled to room temperature and diluted with H₂O (30 mL), acidified withconcentrated HCl solution until pH 3-4. The precipitated solid wascollected by filtration, washed with cold water and dried to afford thedesired product, compound 1505, as an off-white solid. (3.54 g, 84.4%).

To a stirred mixture of concentrated H₂SO₄ (3.6 mL) and fuming HNO₃ (3.1mL, 73.9 mmol), the acid 1505 (2.813 g, 20 mmol) was added at 70-80° C.The reaction mixture was stirred for 6 h at 70° C., and cooled to roomtemperature and then poured into ice-water. The precipitated solid wascollected by filtration, washed with water and dried to afford thedesired product, compound 1506, as a yellow solid (0.795 g, 21.5%).

A mixture of compound 1506 (1.508 g, 8.15 mmol) and SOCl₂ (6 mL) wasrefluxed for 3 h, then concentrated to remove SOCl₂. The residue wasdissolved in CH₂Cl₂ (8 mL). To this solution, Et₃N (1.13 mL) ando-toluidine (1.12 g, 12.23 mmol) was added at 0° C. The resultingmixture was stirred for 2 h at 10° C., concentrated and diluted withwater. The solid was collected by filtration, washed with water andpetroether, dried to afford the desired product, compound 1507, as ayellow solid (1.74 g, 77.6%).

To a stirred mixture of compound 1507 (1.73 g, 6.31 mmol) in MeOH (100mL) and THF (10 mL), 5% Pd/C (0.2 g) was added. The mixture was degassedand back-filled with hydrogen three times. The reaction mixture wasstirred overnight at room temperature and then filtrated. The filtratewas concentrated in vacuo. The solid was dried to afford the desiredproduct, compound 1508, as a pale solid (1.47 g, 95.4%).

Chloroacetyl chloride (1.44 mL, 1.99 mmol) was added to a solution ofcompound 1508 (1.46 g, 5.98 mmol) in acetic acid (20 mL) and thereaction mixture was heated to reflux for 4 h. The reaction mixture wascooled to room temperature and concentrated in vacuo. The residue wasdiluted in DCM (100 mL), washed with saturated NaHCO₃ solution andbrine, dried and concentrated. The residue was purified by flashchromatography eluting with petroether in ethyl acetate (10/1) to affordthe desired product, compound 1509, as a off-white solid (0.48 g,26.7%).

A solution of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (108A) (311 mg,1.19 mmol) and K₂CO₃ (330 mg, 2.39 mmol) in DMF (10 mL) was stirred atroom temperature for 15 min., a solution of compound (1509) (180 mg,1.15 mmol, 1 eq.) in DMF (5 mL) was added dropwise at room temperature.The resulting mixture was stirred 2 h at 80° C. The reaction mixture wasconcentrated in vacuo to remove the organic solvent. The resultingresidue was purified by a silica gel column chromatography to afford thedesired product, compound 1509, (142 mg, 44.9% yield) as a pale yellowsolid.

Compound 1510 (40 mg, 0.076 mmol), Na₂CO₃ (40 mg, 0.38 mmol),Pd(PPh₃)₄(17.6 mg, 0.015 mmol), and 3-fluoro-4-hydroxyphenylboronic acid(15.8 mg, 0.101 mmol) were dissolved in a solution of DMF, ethanol andwater (4 mL/2 mL/2 mL). The resulting mixture was degassed andback-filled with argon three times and then heated to 80° C. for 4 hwith stirring. The reaction mixture was cooled to room temperature,neutralized with 1N HCl solution until pH 7, concentrated in vacuo andextracted with ethyl acetate. The combined organic phases were driedover Na₂SO₄, filtered and concentrated. The residue was purified byflash column chromatography eluting with DCM/MeOH=50/1 to afford thedesired product 1511 (32 mg, 82%).

Example 3: Synthesis of3-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one(Compound 1610) (Method A)

A solution of 2-amino-6-methylbenzoic acid (104) (106.5 g, 705 mmol) inH₂O (200 mL) was cooled to 0-5° C., con. HCl (250 mL) was added slowly.The solution was stirred for 15 min at 0-5° C. A solution of sodiumnitrite (58.4 g, 6.85 mol) in H₂O (120 mL) was added dropwise at 0-5°C., and the resulting mixture was stirred for 30 min. Then abovesolution was added to a solution of KI (351 g, 2.11 mol) in H₂O (200mL), and the resulting mixture was stirred at RT for 16 h. The solutionwas poured into ice water (2000 mL) and extracted with ethyl acetate(3×1000 mL). The combined organic layer was washed with aqueous NaOH(15%, 3×200 mL). The aqueous layer was acidified to PH=1, and extractedwith ethyl acetate (3×1000 mL). The combined organic layer was driedover Na₂SO₄ and filtered. The filtrate was concentrated in vacuo toafford the desired product, 2-iodo-6-methylbenzoic acid (901) (145 g,79% yield) as a yellow solid

To a stirred mixture of 2-iodo-6-methylbenzoic acid (901) (105 g, 400mmol), Pd(OAc)₂ (27 g, 120 mmol) and PPh₃ (63 g 240 mol) in THF (1000mL) at RT, tributyl(vinyl)tin (152 g, 480 mmol) was added. The resultingmixture was heated to reflux overnight. The mixture was allowed to coolto RT, filtered through silica gel (10 g), and then concentrated invacuo. The residue was poured into ice water (1000 mL) and extractedwith ethyl acetate (3×1000 mL). The combined organic layer was washedwith aqueous NaOH (15%, 5×200 mL). The combined aqueous layer wasacidified to PH=1, extracted with ethyl acetate (3×1000 mL). Thecombined organic layer was dried over Na₂SO₄ and filtered. The filtratewas concentrated in vacuo to afford the desired product,2-methyl-6-vinylbenzoic acid (902) (61 g, 95% yield) as a yellow solid.

A mixture of 2-methyl-6-vinylbenzoic acid (902) (56 g, 350 mmol) andthionyl chloride (208 g, 1750 mmol) in toluene (400 mL) was stirred atreflux for 2 h. The mixture was concentrated in vacuo to afford thedesired product, 2-methyl-6-vinylbenzoyl chloride (1601) (63 g, 95%yield) as a yellow oil. The product obtained was used directly in thenext step without purification.

A mixture of o-toluidine (45 g, 420 mmol) and Triethylamine (71 g, 70mmol) in CH₂Cl₂ (300 mL) was stirred for 10 min at RT. To this mixture,2-methyl-6-vinylbenzoyl chloride (1601) (63 g, 35 mmol) was added, andthe resulting mixture was stirred at RT for 30 min. The solution waspoured into water (300 mL) and extracted with CH₂Cl₂ (3×200 mL), driedover Na₂SO₄ and filtered. The filtrate was concentrated in vacuo toafford the crude product. The crude product was suspended in IPE(isopropyl ether) (300 mL), stirred at reflux for 30 min, and thencooled to 0-5° C. The precipitate was collected by filtration andfurther dried in vacuo to afford the desired product,2-methyl-N-o-tolyl-6-vinylbenzamide (1602) (81 g, 80% yield) as a yellowsolid.

To a solution of 2-methyl-N-o-tolyl-6-vinylbenzamide (1602) (80 g, 320mmol) in DMF (250 mL) at RT, NaH (60% in mineral oil, 25.6 g, 640 mmol)was slowly added and the resulting mixture was stirred at RT for 30 min.To this mixture, ethyl chloroacetate (78 g, 640 mmol) was added and theresulting mixture was stirred at RT for 2 h. The solution was pouredinto water (500 mL) and extracted with ethyl acetate (3×200 mL), driedover Na₂SO₄ and filtered. The filtrate was concentrated in vacuo. Thecrude product was suspended in MeOH (160 mL), stirred at reflux for 10min, and then cooled to 0-5° C. The precipitate was collected byfiltration and further dried in vacuo to afford the desired product,ethyl 2-(2-methyl-N-o-tolyl-6-vinylbenzamido) acetate (1603) (67 g, 62%yield) as a white solid.

To a stirred mixture of ethyl 2-(2-methyl-N-o-tolyl-6-vinylbenzamido)acetate (1603) (67 g, 200 mmol) in 1, 4-dioxane (300 mL) and H₂O (100mL) at RT, Osmium tetroxide (20 mg) was added was and stirred at RT for30 min. To this mixture, sodium periodate (86 g, 400 mmol) was added andthe resulting mixture was stirred at RT for 16 h. The reaction mixturewas filtered through silica gel (10 g), the filtrate was extracted withethyl acetate (3×200 mL). The combined organic layers were washed withbrine (100 mL), dried over Na₂SO₄ and filtered. The filtrate wasconcentrated in vacuo and the residue was further dried in vacuo toafford the desired product, ethyl2-(2-formyl-6-methyl-N-o-tolylbenzamido) acetate (1604) (38 g, 57%yield) as a yellow solid.

To a stirred solution of ethyl 2-(2-formyl-6-methyl-N-o-tolylbenzamido)acetate (1604) (38 g, 112 mmol) in EtOH (200 mL) and ethyl acetate (100mL) at RT, cesium carbonate (22 g, 112 mmol) was added. The resultingmixture was degassed and back-filled with argon three times and thenstirred at 50° C. for 5 h. The mixture was allowed to cool to RT,filtered through silica gel (10 g), and the filtrate was concentrated invacuo. The residue was poured into H₂O (200 mL), extracted with ethylacetate (3×200 mL). The combined organic layer was washed with brine (50mL), dried over Na₂SO₄ and filtered. The filtrate was concentrated invacuo. The crude product was suspended in IPE (120 mL), heated to refluxfor 10 min, and then cooled to 0-5° C. The precipitate was collected byfiltration and further dried in vacuo to afford the desired product,ethyl 8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinoline-3-carboxylate(1605) (28 g, 77% yield) as a white solid.

To a stirred solution of lithium aluminum hydride (8.28 g, 218 mol) inanhydrous THF (500 mL) at −78° C. under a nitrogen atmosphere, ethyl8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinoline-3-carboxylate (1605)(28 g, 87 mmol) was slowly added over a 10 min period of time. Theresulting mixture was allowed to warm to −30° C., stirred for 30 min andTLC showed the completion of the reaction. Then the mixture was cooledto −78° C., and water (50 mL) was slowly added. The mixture was allowedto warm to RT, filtered through silica gel (10 g), and the filtrate wasconcentrated in vacuo. The crude product was poured into H₂O (200 mL)and extracted with ethyl acetate (3×200 mL). The combined organic layerwas washed with brine (50 mL), dried over Na₂SO₄ and filtered. Thefiltrate was concentrated in vacuo. The crude product was suspended inethyl acetate (30 mL) and stirred for 10 min. The solid was collected byfiltration and further dried in vacuo to afford the desired product,3-(hydroxymethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one (1606) (22 g,92% yield) as a white solid.

PBr₃ (25.6 g, 95 mmol) was slowly added to a stirred solution of DMF(11.5 g, 158 mol) in acetonitrile (200 mL) at 0° C., and the resultingmixture was stirred at 0 C for 30 min.3-(Hydroxymethyl)-8-methyl-2-o-tolylisoquinolin-1-(2H)-one (1606) (22 g,78.8 mmol) was slowly added. Then the reaction mixture was allowed towarm to RT and stirred for 30 min. Saturated aqueous NaHCO₃ solution (50mL) was slowly added and extracted with ethyl acetate (3×200 mL). Thecombined organic layer was washed with brine, dried over Na₂SO₄ andfiltered. The filtrate was concentrated in vacuo. The crude product wassuspended in IPE (50 mL) and then stirred for 10 min. The precipitatewas collected by filtration and further dried in vacuo to afford thedesired product, 3-(bromomethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one(1607) (21 g, 80% yield) as a white solid.

3-Iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (108A) (10.8 g, 41.4 mmol)and potassium tert-butoxide (4.4 g, 40 mmol) were dissolved in anhydrousDMF (150 mL) and stirred at RT for 30 min.3-(Bromomethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one (1607) (13.7 g,40 mmol) was added. The resulting mixture was stirred at RT for 30 min,poured into ice water (300 mL) and then extracted with ethyl acetate(3×200 mL). The combined organic layer was washed with brine (50 mL),dried over Na₂SO₄ and filtered. The filtrate was concentrated to about100 ml in vacuo, the precipitate was collected by filtration to affordthe first batch of desired product,3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one(1608) (12 g, 60% yield) as a white solid. The filtrate was concentratedin vacuo and the residue was purified by flash column chromatography onsilica gel (2-20% MeOH/DCM) to afford the second batch of desiredproduct,3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one(1608) (6 g, 30% yield) as a white solid.

3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one(1608) (13 g, 24.9 mmol) and3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (1609)(6.6 g, 30mmol) were dissolved in DMF-EtOH-H₂O (120 mL, 40 mL, 40 mL). Pd(OAc)₂(1.684 g, 7.5 mmol), PPh₃ (3.935 g 15 mmol) and Na₂CO₃ (13.25 g 125mmol) were added sequentially. The resulting mixture was degassed andback-filled with argon three times and then stirred at 100° C. for 1 h.The mixture was allowed to cool to RT, filtered through silica gel (10g) and concentrated in vacuo. The residue was purified by flash columnchromatography on silica gel (2-20% MeOH/DCM) to afford the product(1610) (9 g, 76% yield) as a slight yellow solid. Then above product wassuspended in EtOH (100 mL) and heated to reflux for 30 min. The mixturewas allowed to cool to RT, and the solid was collected by filtration.The solid was then suspended in EA (100 mL) and stirred overnight. Theprecipitate was collected by filtration and further dried in vacuo toafford the desired product,3-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one(1610)(8.4 g, 69% yield) as a white solid.

Example 4: Synthesis of3-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o-tolylisoguinolin-1(2H)-one(Compound 1610) (Method B)

3-(3-Methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (1701)(964 mg, 4mmol) and potassium tert-butoxide (0.44 g, 4 mmol) were dissolved inanhydrous DMF (150 mL) and stirred at RT for 30 min.3-(Bromomethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one (1607) (1.37 g,4.0 mmol) was added. The resulting mixture was stirred at RT for 30 min,poured into ice water (30 mL) and then extracted with ethyl acetate(3×50 mL). The combined organic layer was washed with brine (25 mL),dried over Na₂SO₄ and filtered. The filtrate was concentrated in vacuoand the residue was purified by flash column chromatography on silicagel (2-20% MeOH/DCM) to afford the desired product,3-((4-amino-3-(3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one(1702) (1.4 g, 70% yield) as a white solid.

To a solution of3-((4-amino-3-(3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one(1702)(100 mg, 0.2 mmol) in CH₂Cl₂ (20 mL) at −78° C. under a nitrogenatmosphere, BBr₃ (1 mL) was added and the resulting mixture was stirredat −78° C. for 3 h. The mixture was allowed to warm to RT, poured intoice-water (200 mL) and extracted with ethyl acetate (3×50 mL). Thecombined organic layer was washed with brine (20 mL), dried over Na₂SO₄and filtered. The filtrate was concentrated in vacuo and the residue waspurified by flash column chromatography on silica gel (10-50%MeOH/CH₂Cl₂) to afford the desired product,3-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one (1610)(87 mg, 91% yield) as a white solid.

Example 5: Synthesis of(R)-3-((4-amino-3-(3-hydroxybut-1-ynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one(Compound 1802)

3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one(1608)(522 mg, 1 mmol) and (R)-but-3-yn-2-ol (84 mg, 1.2 mmol) were dissolvedin anhydrous THF (40 mL). The mixture was degassed and back-filled withnitrogen three times. Pd(PPh₃)₂Cl₂ (12 mg, 0.1 mmol), CuI (47 mg 0.25mmol) and (i-Pr)₂NH (505 mg, 5 mmol) were added sequentially. Theresulting mixture was degassed and back-filled with argon three timesand then stirred at reflux for 4 h. The mixture was allowed to cool toRT, filtered through silica gel (10 g) and concentrated in vacuo. Theresidue was purified by flash column chromatography on silica gel (2-20%MeOH/DCM) to afford the product, 3(R)-3-((4-amino-3-(3-hydroxybut-1-ynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one(1802) (324 mg, 70% yield) as a slightly yellow solid.

Example 6: Synthesis of3-((6-amino-9H-purin-9-yl)methyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one(Compound 1902)

9H-Purin-6-amine (1901)(540 mg, 4.0 mmol) was dissolved in anhydrous DMF(20 mL). NaH (60% in mineral oil, 160 mg, 4.0 mmol) was added and theresulting mixture was stirred at RT for 30 min.3-(Bromomethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one (1607)(1.37 g,4.0 mmol) was added. The reaction mixture was stirred at RT for 30 min,poured into ice-water (30 mL) and then extracted with ethyl acetate(3×50 mL). The combined organic layer was washed with brine (25 mL),dried over Na₂SO₄ and filtered. The filtrate was concentrated in vacuoand the residue was purified by flash column chromatography on silicagel (2-20% MeOH/DCM) to afford the desired product,3-((6-amino-9H-purin-9-yl)methyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one(1902)(1.1 g, 70% yield) as a white solid.

Example 7: Synthesis of3-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-isopropyl-8-methylisoquinolin-1(2H)-one(Compound 2009)

To a stirred mixture of 2-iodo-6-methylbenzoic acid (901) (105 g, 400mmol), Pd(OAc)₂ (27 g, 120 mmol) and PPh₃ (63 g 240 mol) in THF (1000mL) at RT, tributyl(vinyl)tin (152 g, 480 mmol) was added. The resultingmixture was heated to reflux overnight. The mixture was allowed to coolto RT, filtered through silica gel (10 g), and then concentrated invacuo. The residue was poured into ice water (1000 mL) and extractedwith ethyl acetate (3×1000 mL). The combined organic layer was washedwith aqueous NaOH (15%, 5×200 mL). The combined aqueous layer wasacidified to PH=1, extracted with ethyl acetate (3×1000 mL). Thecombined organic layer was dried over Na₂SO₄ and filtered. The filtratewas concentrated in vacuo to afford the desired product,2-methyl-6-vinylbenzoic acid (902) (61 g, 95% yield) as a yellow solid.

A mixture of 2-methyl-6-vinylbenzoic acid (902) (56 g, 350 mmol) andthionyl chloride (208 g, 1750 mmol) in toluene (400 mL) was stirred atreflux for 2 h. The mixture was concentrated in vacuo to afford thedesired product, 2-methyl-6-vinylbenzoyl chloride (1601) (63 g, 95%yield) as a yellow oil. The product obtained was used directly in thenext step without purification.

Propan-2-amine (2001)(59 g, 1.0 mol) and ethyl chloroacetate (122 g, 1.0mol) were dissolved in toluene (200 mL) and the mixture was stirred atreflux for 2 h. The reaction mixture was allowed to cool to RT, pouredinto ice-water (500 mL) and extracted with ethyl acetate (3×250 mL). Thecombined organic layer was washed with brine (50 mL), dried over Na₂SO₄and filtered. The filtrate was concentrated in vacuo and the residue waspurified by flash column chromatography on silica gel (10-50% EA/PE) toafford the product, ethyl 2-(isopropylamino)acetate (2002) (70 g, 51%yield) as an oil.

Ethyl 2-(isopropylamino)acetate (2002) (14.5 g, 100 mmol) andtriethylamine (200 g, 200 mmol) were dissolved in CH₂Cl₂ (300 mL) andthe mixture was stirred for 10 min at RT. 2-Methyl-6-vinylbenzoylchloride (1601) (18 g, 100 mmol) was added, and the resulting mixturewas stirred at RT for 30 min. The reaction mixture was poured into water(300 mL) and extracted with CH₂Cl₂ (3×200 mL). The combined organiclayer was washed with brine (50 mL), dried over Na₂SO₄ and filtered. Thefiltrate was concentrated in vacuo to afford the crude product. Thecrude product was suspended in IPE (isopropyl ether) (300 mL), stirredat reflux for 30 min, and then cooled to 0-5° C. The precipitate wascollected by filtration and further dried in vacuo to afford the desiredproduct, ethyl 2-(N-isopropyl-2-methyl-6-vinylbenzamido)acetate (2003)(14.5 g, 50% yield) as a yellow solid.

To a stirred solution of ethyl2-(N-isopropyl-2-methyl-6-vinylbenzamido)acetate (2003) (14.0 g, 48.0mmol) in 1,4-dioxane (100 mL) and H₂O (30 mL), Osmium tetroxide (20 mg)was added and the resulting mixture was stirred at RT for 30 min. Tothis mixture, sodium periodate (22 g, 100 mmol) was added and thenstirred at RT for 16 h. The reaction mixture was filtered through silicagel (10 g), the filtrate was extracted with ethyl acetate (3×200 mL).The combined organic layer was washed with brine (50 mL), dried overNa₂SO₄ and filtered. The filtrate was concentrated in vacuo and theresidue was further dried in vacuo to afford the desired product, ethyl2-(2-formyl-N-isopropyl-6-methylbenzamido)acetate (2004) (8.33 g, 57%yield) as a yellow solid.

To a stirred solution of ethyl2-(2-formyl-N-isopropyl-6-methylbenzamido)acetate (2004) (8.3 g, 28.0mmol) in EtOH (100 mL) and ethyl acetate (50 mL) at RT, cesium carbonate(5.9 g, 30 mmol) was added. The resulting mixture was degassed andback-filled with argon three times and then stirred at 50° C. for 5 h.The mixture was allowed to cool to RT, filtered through silica gel (10g), and the filtrate was concentrated in vacuo. The residue was pouredinto H₂O (200 mL), extracted with ethyl acetate (3×200 mL). The combinedorganic layer was washed with brine (50 mL), dried over Na₂SO₄ andfiltered. The filtrate was concentrated in vacuo. The crude product wassuspended in IPE (120 mL), stirred at reflux for 10 min, and then cooledto 0-5° C. The precipitate was collected by filtration and further driedin vacuo to afford the desired product, ethyl2-isopropyl-8-methyl-1-oxo-1,2-dihydroisoquinoline-3-carboxylate (2005)(5.35 g, 70% yield) as a white solid.

To a stirred solution of lithium aluminum hydride (2.88 g, 76 mol) inanhydrous THF (200 mL) at −78° C. under a nitrogen atmosphere, ethyl2-isopropyl-8-methyl-1-oxo-1,2-dihydroisoquinoline-3-carboxylate (2005)(5.2 g, 19 mmol) was slowly added over a 10 min period of time. Theresulting mixture was allowed to warm to −30° C., stirred for 30 min andTLC showed the completion of the reaction. Then the mixture was cooledto −78° C., and water (50 mL) was slowly added. The mixture was allowedto warm to RT, filtered through silica gel (10 g), and the filtrate wasconcentrated in vacuo. The crude product was poured into H₂O (200 mL)and extracted with ethyl acetate (3×200 mL). The combined organic layerwas washed with brine (50 mL), dried over Na₂SO₄ and filtered. Thefiltrate was concentrated in vacuo. The crude product was suspended inethyl acetate (30 mL) and stirred for 10 min. The solid was collected byfiltration and further dried in vacuo to afford the desired product,3-(hydroxymethyl)-2-isopropyl-8-methylisoquinolin-1(2H)-one (2006) (3.51g, 80% yield) as a white solid.

To a solution of3-(hydroxymethyl)-2-isopropyl-8-methylisoquinolin-1(2H)-one (2006) (1.61g, 7.0 mmol) in CH₂Cl₂, PPh₃ (3.67 g, 14.0 mmol) was added and themixture was stirred at RT for 30 min. The mixture was cooled to 0° C.,and CBr₄ (4.64 g, 14.0 mmol) was added in portions. The resultingmixture was stirred from 0° C. to RT for 30 min, and then concentratedin vacuo. The crude product was purified by flash column chromatographyon silica gel (30-50% EA/PE) to afford the desired product,3-(bromomethyl)-2-isopropyl-8-methylisoquinolin-1(2H)-one (2007) (1.65g, 80% yield) as a white solid.

A mixture of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (108A) (1.3 g, 5mmol) and potassium tert-butoxide (0.55 g, 5 mmol) in anhydrous DMF (20mL) was stirred at RT for 30 min and then3-(bromomethyl)-2-isopropyl-8-methylisoquinolin-1(2H)-one (2007) (1.47g, 5 mmol) was added. The resulting mixture was stirred at RT for 30min, poured into ice-water (30 mL) and then extracted with ethyl acetate(3×50 mL). The combined organic layer was washed with brine (25 mL),dried over Na₂SO₄ and filtered. The filtrate was concentrated in vacuo,and the residue was purified by flash column chromatography on silicagel (2-20% MeOH/DCM) to afford the desired product,3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-isopropyl-8-methylisoquinolin-1(2H)-one(2008) (1.66 g, 70% yield) as a white solid.

To a stirred mixture of3-((4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-isopropyl-8-methylisoquinolin-1(2H)-one(2008) (95 mg, 0.2 mmol) and3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (66 mg, 0.3 mmol)in DMF-EtOH-H₂O (3:1:1, 20 mL), Pd(OAc)₂ (16 mg, 0.075 mmol), PPh₃ (39.3mg 0.15 mmol) and Na₂CO₃ (132 mg, 1.25 mmol) were added sequentially.The resulting mixture was degassed and back-filled with argon threetimes and then stirred at 100° C. for 1 h. The mixture was allowed tocool to RT, filtered through silica gel (10 g) and concentrated invacuo. The residue was purified by flash column chromatography on silicagel (2-20% MeOH/DCM) to afford the product,3-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-isopropyl-8-methylisoquinolin-1(2H)-one(2009) (53 mg, 61% yield) as a slightly yellow solid.

Example 8: Synthesis of7-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-4-methyl-6-o-tolyl-1,6-naphthyridin-5(6H)-one(Compound 2115)

To a mixture of ethyl 2-cyanoacetate (2101) (45.2 g, 400 mmol) andacetone (46.4 g, 800 mmol) in glacial acetic acid (50 mL), piperidine (2mL, 20 mmol) was added and the resulting mixture stirred at reflux for24 h. The reaction mixture was allowed to cool to RT, and thenconcentrated in vacuo. The residue was diluted with water (200 mL) andextracted with ethyl acetate (3×200 mL). The combined organic layer waswashed with brine (50 mL), dried over Na₂SO₄ and filtered. The filtratewas concentrated in vacuo and the residue was purified by flash columnchromatography on silica gel (0-2% EA/PE) to afford the desired product,ethyl 2-cyano-3-methylbut-2-enoate (2102) (49.6 g, 81% yield) as a whitesolid.

To a solution of ethyl 2-cyano-3-methylbut-2-enoate (2102) (43.6 g, 285mol) in absolute EtOH (300 mL), N,N-dimethylformamide dimethyl acetal(37.3 g, 313 mmol) was added dropwise and the resulting mixture wasstirred at reflux 6 h. The mixture was allowed to cool to RT,concentrated in vacuo to afford the crude desired product, ethyl2-cyano-5-(dimethylamino)-3-methylpenta-2,4-dienoate (2103) (39.8 g, 67%yield) as a yellow solid.

Ethyl 2-cyano-5-(dimethylamino)-3-methylpenta-2,4-dienoate (2103) (30.8g, 148 mmol) was dissolved in AcOH (120 mL) and the mixture was stirredat 40° C. A solution of 45% HBr—AcOH (120 mL) was added dropwise, andthen the mixture was stirred at 55° C. for 2 h. The mixture was allowedto cool to RT, poured onto ice, neutralized with solid Na₂CO₃, andextracted with ethyl acetate (3×150 mL). The combined organic layer waswashed with brine (50 mL), dried over Na₂SO₄ and filtered. The filtratewas concentrated in vacuo and the residue was purified by flash columnchromatography on silica gel (5-20% EA/PE) to afford the desiredproduct, ethyl 2-bromo-4-methylnicotinate (2104) (17.6 g, 49% yield) asa yellow oil.

To a solution of ethyl 2-bromo-4-methylnicotinate (2104) (12.8 g, 52mmol) in 1,4-dioxane (15 mL), a solution of NaOH (8.0 g, 200 mmol) inH₂O (15 mL) was added and the resulting mixture was stirred at refluxfor 12 h. The mixture was allowed to cool to RT, diluted with H₂O,washed with ethyl acetate (3×30 mL). The aqueous layer was acidifiedwith concentrated hydrochloric acid to pH=1, and extracted with ethylacetate (3×50 mL). The combined organic layer was washed with brine (25mL), dried over Na₂SO₄ and filtered. The filtrate was concentrated invacuo to afford the desired product, 2-bromo-4-methylnicotinic acid(2105) (9.7 g, 85% yield) as a white solid.

To a solution of 2-bromo-4-methylnicotinic acid (2105) (13 g, 60 mmol)and DMF (3 drops) in CH₂Cl₂ (150 mL), oxalyl chloride (11.4 g, 90 mmol)was added dropwise and the resulting mixture was stirred at RT for 2 h.The reaction mixture was concentrated in vacuo to afford the desiredproduct, 2-bromo-4-methylnicotinoyl chloride (2106) (13.4 g, 95% yield)as a yellow oil. The product obtained was used directly in the next stepwithout further purification.

o-Toluidine (7.7 g, 72 mmol) and triethylamine (9.1 g, 90 mmol) weredissolved in CH2Cl2 (100 mL) and stirred for 10 min at RT.2-Bromo-4-methylnicotinoyl chloride (2106) (13.4 g, 57 mmol) was added,and the resulting mixture was stirred at RT for 1 h. The mixture waspoured into water (200 mL) and extracted with CH₂Cl₂ (3×50 mL). Thecombined organic layer was washed with brine (20 mL), dried over Na₂SO₄and filtered. The filtrate was concentrated in vacuo to afford the crudeproduct. The crude product was suspended in IPE (isopropyl ether) (50mL). The mixture was stirred at reflux for 30 min and then was cooled to0-5° C. The precipitate was collected by filtration and further dried invacuo to afford the desired product,2-bromo-4-methyl-N-o-tolylnicotinamide (2107) (13 g, 75% yield) as ayellow solid.

To a solution of 2-bromo-4-methyl-N-o-tolylnicotinamide (2107) (13 g, 43mmol) and tributyl(vinyl)tin (16.4 g, 52 mmol) in THF (200 mL) under anitrogen atmosphere, Pd(OAc)₂ (2.9 g, 13 mmol) and PPh₃ (6.8 g, 26 mol)were added. The resulting mixture was stirred at reflux for 16 h. Themixture was then allowed to cool to RT, filtered through silica gel (10g), and concentrated in vacuo. The residue was poured into water (200mL), extracted with ethyl acetate (3×50 mL). The combined organic layerwas washed with brine (30 mL), dried over Na₂SO₄ and filtered. Thefiltrate was concentrated in vacuo and the residue was purified by flashcolumn chromatography on silica gel (20-50% EA/PE) to afford the desiredproduct, 4-methyl-N-o-tolyl-2-vinylnicotinamide (2108) (8.7 g, 80%yield) as a yellow solid.

To a stirred solution of 4-methyl-N-o-tolyl-2-vinylnicotinamide (2108(8.1 g, 32 mmol) in DMF (50 mL) at RT, NaH (60% in mineral oil, 2.6 g,65 mmol) was added slowly and the resulting mixture was stirred at RTfor 30 min. Ethyl chloroacetate (78 g, 640 mmol) was added dropwise tothis mixture at RT, and stirred for 2 h. The solution was poured intowater (300 mL) and extracted with ethyl acetate (3×80 mL). The combinedorganic layer was washed with brine (25 mL), dried over Na₂SO₄ andfiltered. The filtrate was concentrated in vacuo. The crude product wassuspended in MeOH (60 mL) and stirred at reflux for 10 min. The mixturewas then cooled to 0-5° C. The precipitate was collected by filtrationand further dried in vacuo to afford the desired product, ethyl2-(4-methyl-N-o-tolyl-2-vinylnicotinamido) acetate (2109) (6.3 g, 58%yield) as a white solid.

To a solution of ethyl 2-(4-methyl-N-o-tolyl-2-vinylnicotinamido)acetate (2109) (6.1 g, 18 mmol) in 1, 4-dioxane (90 mL) and H₂O (30 mL)at RT, Osmium tetroxide (5 mg) was added and the resulting mixture wasstirred for 30 min. Sodium periodate (7.7 g, 36 mmol) was added and themixture was stirred at RT for 16 h. The mixture was filtered throughsilica gel (5 g), and the filtrate was extracted with ethyl acetate(3×50 mL). The combined organic layer was washed with brine (25 mL),dried over Na₂SO₄ and filtered. The filtrate was concentrated in vacuoand the residue was further dried under reduced pressure to afford thedesired product, ethyl2-(2-formyl-4-methyl-N-o-tolylnicotinamido)acetate (2110) (4.4 g, 72%yield) as a yellow solid.

To a stirred solution of ethyl2-(2-formyl-4-methyl-N-o-tolylnicotinamido)acetate (2110) (4.4 g, 13mmol) in EtOH (30 mL) and ethyl acetate (10 mL) at RT, cesium carbonate(4.3 g, 13 mmol) was added. The resulting mixture was degassed andback-filled with argon three times and then stirred at 50° C. for 5 h.The mixture was allowed to cool to RT, filtered through silica gel (5g), and the filtrate was concentrated in vacuo. The residue was pouredinto H₂O (200 mL), extracted with ethyl acetate (3×50 mL). The combinedorganic layer was dried over Na₂SO₄ and filtered. The filtrate wasconcentrated in vacuo. The crude product was suspended in IPE (30 mL),stirred at reflux for 10 min, and then cooled to 0-5° C. The precipitatewas collected by filtration and further dried in vacuo to afford thedesired product, ethyl4-methyl-5-oxo-6-o-tolyl-5,6-dihydro-1,6-naphthyridine-7-carboxylate(2111) (3.0 g, 72% yield) as a white solid.

To a stirred solution of lithium aluminum hydride (0.86 g, 23 mol) inanhydrous THF (100 mL) at −78° C. under a nitrogen atmosphere, asolution of ethyl4-methyl-5-oxo-6-o-tolyl-5,6-dihydro-1,6-naphthyridine-7-carboxylate(2111) (2.9 g, 9.0 mmol) in anhydrous THF (20 mL) was added dropwise.The resulting mixture was allowed to warm to −10° C., stirred for 30 minand TLC showed the completion of the reaction. Then the mixture wascooled to −78° C., and water (50 mL) was slowly added. The mixture wasallowed to warm to RT, filtered through silica gel (5 g), and thefiltrate was concentrated in vacuo. The crude product was poured intoH₂O (100 mL) and extracted with ethyl acetate (3×50 mL). The combinedorganic layer was washed with brine (25 mL), dried over Na₂SO₄ andfiltered. The filtrate was concentrated in vacuo. The crude product wassuspended in ethyl acetate (10 mL) and stirred for 10 min. The solid wascollected by filtration and further dried in vacuo to afford the desiredproduct, 7-(hydroxymethyl)-4-methyl-6-o-tolyl-1,6-naphthyridin-5(6H)-one(2112) (2.1 g, 83% yield) as a white solid.

To a solution of7-(hydroxymethyl)-4-methyl-6-o-tolyl-1,6-naphthyridin-5(6H)-one (2112)(1.96 g, 7.0 mmol) in CH₂Cl₂, PPh₃ (3.67 g, 14.0 mmol) was added andstirred at RT for 30 min. CBr₄ (4.64 g, 14.0 mmol) was added to themixture in portions at 0° C. The resulting mixture was allowed to warmto RT, stirred for 30 min, and concentrated in vacuo. The residue waspurified by flash column chromatography on silica gel (30-50% EA/PE) toafford the desired product,7-(bromomethyl)-4-methyl-6-o-tolyl-1,6-naphthyridin-5(6H)-one (2113)(1.92 g, 80% yield) as a white solid.

A mixture of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (108A) (1.08 g,4.14 mmol) and potassium tert-butoxide (0.44 g, 4.0 mmol) in anhydrousDMF (50 mL) was stirred at RT for 30 min and then7-(bromomethyl)-4-methyl-6-o-tolyl-1,6-naphthyridin-5(6H)-one (2113)(1.37 g, 4.0 mmol) was added. The resulting mixture was stirred at RTfor 30 min, poured into ice water (300 mL) and then extracted with ethylacetate (3×100 mL). The combined organic layer was washed with brine (30mL), dried over Na₂SO₄ and filtered. The filtrate was concentrated invacuo and the residue was purified by flash column chromatography onsilica gel (0-2% MeOH/DCM) to afford the desired product,7-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-4-methyl-6-o-tolyl-1,6-naphthyridin-5(6H)-one(2114) (1.07 g, 50% yield) as a white solid.

To a stirred mixture of7-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-4-methyl-6-o-tolyl-1,6-naphthyridin-5(6H)-one(2114) (1.05 g, 2.0 mmol) and 3-hydroxyphenylboronic acid (0.33 g, 2.4mmol) in DMF-EtOH-H₂O (3:1:1, 50 mL), Pd(OAc)₂ (0.14 g, 0.60 mmol), PPh₃(0.31 g, 1.2 mmol) and Na₂CO₃ (1.06 g, 10.0 mmol) were addedsequentially. The resulting mixture was degassed and back-filled withargon three times and then stirred at 80° C. for 1 h. The mixture wasallowed to cool to RT, filtered through silica gel (5 g) andconcentrated in vacuo. The residue was purified by flash columnchromatography on silica gel (2-5% MeOH/DCM) to afford the desiredproduct,7-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-4-methyl-6-o-tolyl-1,6-naphthyridin-5(6H)-one(2115) (0.68 g, 69% yield) as a slight yellow solid. Then the productwas dissolved in EtOH (5 mL), and stirred at reflux for 30 min. Thesolution was allowed to cool to RT and the solid was collected byfiltration. The solid was then suspended in ethyl acetate (5 mL), andstirred at RT for 16 h. The precipitate was collected by filtration andfurther dried in vacuo to afford the desired product,7-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-4-methyl-6-o-tolyl-1,6-naphthyridin-5(6H)-one(2115) (0.59 g, 60% yield) as a white solid.

Example 9:3-((4-amino-3-(3-fluoro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o-tolyl-3,4-dihydroisoquinolin-1(2H)-one(Compound 2208)

2-Iodo-6-methyl-N-o-tolylbenzamide (2201)(1.5 g, 4.27 mmol), which hasbeen prepared from the reaction of compound 902 and 2-methyl aniline,and allyltributyl tin (2.10 g, 1.5 mmol) were dissolved in anhydrous DMF(12 mL). The solution was degassed and back-filled with argon (threetimes). Pd(PPh₃)₄(148 mg, 0.13 mmol) was added. The reaction mixture wasdegassed and back-filled with argon (three times), and then stirred at90° C. for 16 h. The mixture was allowed to cool to RT, partitionedbetween ethyl acetate and H₂O. The organic layer was washed with brine,dried over Na₂SO₄ and filtered. The filtrate was concentrated in vacuoand the residue was purified by column chromatography on silica geleluting with EtOAc and Hexanes to afford the desired product,2-allyl-6-methyl-N-o-tolylbenzamide (2202) (1.1 g, 95% yield).

2-Allyl-6-methyl-N-o-tolylbenzamide (2202) (800 mg, 3.01 mmol) wasdissolved in anhydrous dichloromethane (20 mL). mCPBA (70%, 1.11 g, 4.52mmol) was added and the resulting mixture was stirred at RT for 24 h.Na₂SO₃ (1.0 g) was added and stirred for 1 h. The mixture waspartitioned between EtOAc and water. The organic layer was washed withbrine, dried over Na₂SO₄ and filtered. The filtrate was concentrated invacuo, and the residue was purified by column chromatography on silicagel eluting with EtOAc and Hexanes to afford the desired product,2-methyl-6-(oxiran-2-ylmethyl)-N-o-tolylbenzamide (2203)(660 mg, 83%yield).

2-Methyl-6-(oxiran-2-ylmethyl)-N-o-tolylbenzamide (2203) (860 mg, 3.06mmol) was dissolved in anhydrous DMF (15 mL) and cooled to 0° C. underan argon atmosphere. NaH (60% in mineral oil, 245 mg, 6.12 mmol) wasadded in portions and the resulting mixture was stirred at 0° C. for 3h. H₂O (30 mL) was slowly added and the mixture was extracted with EtOAc(3×25 mL). The combined organic layers were washed with brine, driedover Na₂SO₄ and filtered. The filtrate was concentrated in vacuo, andthe residue was purified by column chromatography on silica gel elutingwith EtOAc and Hexanes to afford the desired product,3-(hydroxymethyl)-8-methyl-2-o-tolyl-3,4-dihydroisoquinolin-1(2H)-one(2204) (435 mg, 51% yield).

3-(Hydroxymethyl)-8-methyl-2-o-tolyl-3,4-dihydroisoquinolin-1(2H)-one(2204) (430 mg, 1.53 mmol) was dissolved in anhydrous dichloromethane(25 mL) and cooled to 0° C. under an argon atmosphere. PPh₃ (600 mg,2.29 mmol) and CBr₄ (761 mg, 2.29 mmol) were added sequentially and theresulting mixture was stirred from 0° C. to RT for 16 h. The reactionmixture was partitioned between EtOAc and water. The organic layer waswashed with brine, dried over Na₂SO₄ and filtered. The filtrate wasconcentrated in vacuo, and the residue was purified by columnchromatography on silica gel eluting with EtOAc and Hexanes to affordthe desired product,3-(bromomethyl)-8-methyl-2-o-tolyl-3,4-dihydroisoquinolin-1(2H)-one(2205)(480 mg, 91% yield).

3-(Bromomethyl)-8-methyl-2-o-tolyl-3,4-dihydroisoquinolin-1(2H)-one(2205) (387 mg, 1.12 mmol) and3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (108A)(440 mg, 1.69 mmol)were dissolved in anhydrous DMF (20 mL). K₂CO₃ (309 mg, 2.24 mmol) wasadded and the resulting mixture was stirred at 50° C. for 3 h. Thereaction mixture was partitioned between EtOAc and water. The organiclayer was washed with brine, dried over Na₂SO₄ and filtered. Thefiltrate was concentrated in vacuo, and the residue was purified bycolumn chromatography on silica gel eluting with methanol anddichloromethane to afford the desired product,3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o-tolyl-3,4-dihydroisoquinolin-1(2H)-one(2206) (100 mg, 17% yield).

3-((4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o-tolyl-3,4-dihydroisoquinolin-1(2H)-one(2206) (100 mg, 0.11 mmol) and 3-fluoro-5-hydroxyphenylboronic acid(2207) (36 mg, 0.23 mmol) were dissolved in DME (4 mL). The solution wasdegassed and back-filled with argon (three times). Pd(PPh₃)₄ (6.4 mg,5.5 μmol) and aqueous Na₂CO₃ solution (1.0M, 0.44 mL, 0.44 mmol) wereadded sequentially. The reaction mixture was degassed and back-filledwith argon (three times), and then stirred at 80° C. for 24 h. Themixture was allowed to cool to RT, partitioned between ethyl acetate andbrine. The organic layer was dried over Na₂SO₄, filtered andconcentrated in vacuo. The residue was purified by column chromatographyon silica gel eluting with methanol and dichloromethane to afford thedesired product,3-((4-amino-3-(3-fluoro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o-tolyl-3,4-dihydroisoquinolin-1(2H)-one(2208) (12 mg, 22% yield).

Example 10: Synthesis of6-((4-amino-3-(3-fluoro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-methyl-5-o-tolylisothiazolo[5,4-d]pyrimidin-4(5H)-one(Compound 2313)

A solution of compound 2302 (24.9 g, 0.19 mol) in CH₃CN (50 mL) wasadded to a solution of compound 2301 (30 g, 0.19 mol) in at 0° C. Themixture was stirred for 1 hour at room temperature and poured into 500mL of water. The reaction mixture was allowed to stand for 1 h. Thesolid was precipitated out of the solution, collected by filtration,washed with water and dried to afford the desired compound 2303 as ared-orange solid (50 g, 85.5%).

To a solution of compound 2303 (54 g, 0.175 mol) in ethyl acetate (200mL), a solution of Br₂ (56 g, 0.35 mol) in ethyl acetate (50 mL) wasadded dropwise at room temperature. The resulting mixture was stirredfor 3 h at room temperature. The solid was collected by filtration,washed with ethyl acetate to afford the desired product, compound 2304(40 g, 74.6%).

A mixture of compound 2304 (40 g, 0.13 mol) and saturated Na₂CO₃solution (10 mL) in DMF (100 mL) was heated for 5 h at 80° C. Thereaction mixture was cooled to room temperature and 1 L of water wasadded. The solid was collected by filtration and dried to afford thedesired product, compound 2305 (16 g, 66%).

A mixture of compound 2305 (16 g, 0.086 mol), NaOH (6.88 g, 0.172 mol)in water (50 mL) was heated for 4 h at reflux. The reaction mixture wascooled to room temperature and acidified with 1N HCl solution untilPH=3-4. The solid was collected by filtration and dried to afford thedesired product, compound 2306 (12 g, 88%). A mixture of compound 2306(1 g, 0.0063 mol) in SOCl₂ (15 mL) was stirred for 5 h at reflux, thereaction mixture was cooled to room temperature and concentrated toremove excess SOCl₂. Anhydrous toluene (30 mL) was added to the residueand concentrated. This process was repeated twice to remove the residueof SOCl₂.

The crude compound 2307 was dissolved in dry toluene (5 mL).2-Methyl-aniline (2 g, 0.0187 mol) was added to the above solution. Theresulting mixture was heated for 1 h at reflux, cooled to roomtemperature and filtered. The filtrate was concentrated to dryness andpartitioned between ethyl acetate and brine. The aqueous phase wasextracted with ethyl acetate. The combined organic phases were washedwith brine, dried with MgSO₄ and filtered. The residue was purified byflash chromatography eluting with petroether ethyl acetate from 50:1 to5:1 to afford the desired product, compound 2308 (600 mg, 38.55%).

A solution of compound 2308 (600 mg, 2.43 mmol) and pyridine (0.78 mL)in DCM (30 mL) was stirred for 10 min at 0° C. Chloroacetyl chloride(423 mg, 3.74 mmol) was added. The reaction mixture was stirred for 2 hand quenched with water. The organic phase was separated and washed withwater, brine, dried, filtered and concentrated to afford the desiredproduct, compound 2309 (700 mg, 89%).

A mixture of compound 2309 (600 mg, 1.85 mmol) and POCl₃ (10 mL) washeated overnight at 120-130° C. (oil bath) in a sealed tube. Thereaction mixture was cooled to room temperature and concentrated. Theresidue was partitioned between ethyl acetate and water, and thenbasified with saturated Na₂CO₃ solution until PH 7-8. The organic layerwas separated, washed with brine, dried over MgSO₄ and concentrated. Theresidue was purified by flash chromatography eluting with petroether inethyl acetate (PE/EA=20/1) to afford the desired product, compound 2310as a yellow powder (300 mg, 53.0%).

tert-BuOK (28.7 mg, 0.256 mmol) was added to a solution of compound 2311(76 mg, 0.295 mmol) in dry DMF (3 mL) at RT. The reaction mixture wasstirred for 30 min., and a solution of compound 2310 (60 mg, 0.196 mmol)in DMF (2 mL) was added dropwise. The resulting mixture was stirred for2 h and concentrated. The residue was purified by flash chromatographyeluting with (DCM/MeOH=50/1) to afford the desired product 2312 asoff-white solid (65 mg, 62.7%).

To a solution of compound 2312 (40 mg, 0.076 mmol) in dry DCM (10 mL),BBr₃ (190.4 mg, 0.76 mmol) was added dropwise at −78° C., then thereaction mixture was allowed to warm to room temperature and stirred forovernight. The reaction mixture was poured into ice-water, basified withsaturated NaHCO₃ solution until PH 8-9 and extracted with DCM. Thecombined organic phases were dried over MgSO₄, filtered andconcentrated. The residue was purified by flash chromatography elutingwith DCM/MeOH=30/1 to afford the desired product, compound 2313 (15 mg,38.5%).

Example 11: Synthesis of2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methyl-3-o-tolylthieno[2,3-d]pyrimidin-4(3H)-one(Compound 2407)

To a stirred solution of 2-amino-4-methylthiophene-3-carboxylic acid(2401) (2.4 g, 15.2 mmol) in THF (50 mL), a solution of triphosgene (9.0g, 30 mmol, 2 eq.) in THF (10 mL) was added slowly dropwise at 0° C. in20 min. The resulting mixture was stirred at 0° C. for 2 h. The reactionmixture was quenched with water (20 mL) at 0° C., and then concentratedin vacuo to remove the organic solvent. A brown solid was precipitatedout of solution. The solid was collected by filtration, washed withwater (5 mL×2) and dried in vacuo to afford the desired product5-methyl-1H-thieno[2,3-d][1,3]oxazine-2,4-dione (2402) (2.5 g, 89.3%yield) as a brown solid.

To a stirred solution of o-toluidine, (1.4 g, 12.8 mmol, 1.2 eq) in dryTHF (20 mL), n-BuLi (2.5N, 7.7 mL, 19.3 mmol, 1.8 eq.) was added slowlydropwise at −40° C. under argon atmosphere in 30 min. The resultingmixture was stirred at −40° C. for another 30 min. A solution of5-methyl-1H-thieno[2,3-d][1,3]oxazine-2,4-dione (2402) (1.95 g, 10.7mmol, 1 eq) in dry THF (50 mL) was added slowly dropwise at −40° C. in20 min. The reaction mixture was stirred at −40° C. for 1 h and thenallowed to warm to room temperature for overnight. The reaction mixturewas quenched with water (20 mL) at 0° C., and then neutralized withconcentrated HCl solution until pH 8-9. The mixture was concentrated invacuo to remove the organic solvent. The residue was extracted withethyl acetate (25 mL×3). The combined organic phases were washed withbrine (10 mL), dried over MgSO₄ and concentrated in vacuo. The resultingresidue was purified by a silica gel column chromatography using 5% to20% ethyl acetate in petroether as an eluent to afford the desiredproduct 2-amino-4-methyl-N-o-tolylthiophene-3-carboxamide (2403) (0.74g, 28.1% yield) as a yellow solid.

To a stirred solution of2-amino-4-methyl-N-o-tolylthiophene-3-carboxamide (2403) (740 mg, 3mmol) and pyridine (406.8 mg, 3.6 mmol, 1.2 eq) in dry DCM (20 mL),2-chloroacetyl chloride (284.8 mg, 3.6 mmol, 1.2 eq.) was added slowlydropwise at 0° C. in 30 min. The resulting mixture was stirred at 0° C.for 2 h. The reaction mixture was quenched with water (20 mL) at 0° C.and extracted with DCM. The combined organic phases were washed with 1Nof HCl solution (10 mL), brine (10 mL), dried over MgSO₄ andconcentrated in vacuo to afford the desired product2-(2-chloroacetamido)-4-methyl-N-o-tolylthiophene-3-carboxamide (2404)(950 mg, 98.1% yield) as a yellow solid.

A mixture of2-(2-chloroacetamido)-4-methyl-N-o-tolylthiophene-3-carboxamide (2404)(1.07 g, 3.32 mmol) and POCl₃ (25 mL) was stirred overnight in a sealedtube at 120° C. The reaction mixture was concentrated in vacuo to removeexcess POCl₃. The residue was partitioned between DCM (30 mL) andsaturated NaHCO₃ solution (10 mL). The organic layer was separated andwashed with sat. NaHCO₃ (10 mL), brine (10 mL), dried over MgSO₄ andconcentrated in vacuo. The resulting residue was purified by a silicagel column chromatography to afford the desired product2-(chloromethyl)-5-methyl-3-o-tolylthieno[2,3-d]pyrimidin-4(3H)-one(2405) (760 mg, 75.2% yield) as a yellow solid.

A solution of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (108A) (314.3mg, 1.2 mmol, 1.5 eq) and t-BuOK (155 mg, 1.38 mmol, 1.2 eq) in DMF (10mL) was stirred at room temperature for 15 min., a solution of2-(chloromethyl)-5-methyl-3-o-tolylthieno[2,3-d] pyrimidin-4(3H)-one(2405) (350 mg, 1.15 mmol, 1 eq.) in DMF (5 mL) was added dropwise atroom temperature. The resulting mixture was stirred at room temperaturefor 2 h. The reaction mixture was concentrated in vacuo to remove theorganic solvent. The resulting residue was purified by a silica gelcolumn chromatography to afford the desired product2-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methyl-3-o-tolylthieno[2,3-d]pyrimidin-4(3H)-one(2406) (250 mg, 41.1% yield) as a yellow solid.

2-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methyl-3-o-tolylthieno[2,3-d]pyrimidin-4(3H)-one(2406) (50 mg, 0.092 mmol), PPh₃ (14.5 mg, 0.056 mmol, 0.6 eq) and3-fluoro-5-hydroxyphenylboronic acid (2207) (17.2 mg, 0.11 mmol, 1.2 eq)were dissolved in a solution of DMF, ethanol and water (5 mL/2 mL/2 mL).To this mixture Pd(OAc)₂ (4.14 mg, 0.018 mmol, 0.2 eq) and sodiumcarbonate (48.7 mg, 0.46 mmol, 5 eq) were added sequentially. Theresulting mixture was degassed and back-filled with argon three timesand then heated to 80° C. for 0.5 h with stirring. The reaction mixturewas concentrated in vacuo to remove the organic solvent. The resultingresidue was purified by a silica gel column chromatography to afford thedesired product2-((4-amino-3-(3-fluoro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methyl-3-o-tolylthieno[2,3-d]pyrimidin-4(3H)-one(2407)(22.8 mg, 48.2% yield) as a yellow solid.

Example 12: Synthesis of8-methyl-3-((methyl(9H-purin-6-yl)amino)methyl)-2-o-tolylisoquinolin-1(2H)-one

3-(Bromomethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one (342 mg, 1.0mmol) (1607) was dissolved in methylamine solution (100 mL) and stirredfor 2 h. The mixture was poured into ice-water (200 mL) and extractedwith ethyl acetate (3×50 mL). The combined organic layer was washed withbrine (20 mL), dried over Na₂SO₄ and filtered. The filtrate wasconcentrated in vacuo to afford the desired product,8-methyl-3-((methylamino)methyl)-2-o-tolylisoquinolin-1(2H)-one (4001)(250 mg, 86% yield) as a yellow solid. The product obtained was useddirectly in the next step without purification.

8-Methyl-3-((methylamino)methyl)-2-o-tolylisoquinolin-1(2H)-one (233 mg,0.8 mmol) (4001) and 6-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine(4002) (238 mg, 1.0 mmol) were dissolved in EtOH (50 mL) and theresulting mixture was stirred at reflux for 2 h. The mixture was allowedto cool to RT, and concentrated in vacuo. The residue was purified byflash column chromatography on silica gel (2-20% MeOH/DCM) to afford theproduct,8-Methyl-3-((methyl(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)amino)methyl)-2-o-tolylisoquinolin-1(2H)-one(4003) (200 mg, 51% yield) as a slight yellow solid.

8-Methyl-3-((methyl(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)amino)methyl)-2-o-tolylisoquinolin-1(2H)-one(4003) (180 mg 0.36 mmol) was dissolved in MeOH (HCl) (50 mL) and themixture was stirred at RT for 2 h. Aqueous NaHCO₃ solution was added tothe reaction mixture and the pH was adjusted to 9. The mixture wasfiltered and the filtrate was concentrated in vacuo to afford thedesired product,8-methyl-3-((methyl(9H-purin-6-yl)amino)methyl)-2-o-tolylisoquinolin-1(2H)-one(4004) (80 mg, 54% yield) as a yellow solid.

Example 13: Synthesis of3-(1-(9H-purin-6-ylamino)ethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one

To a stirred solution of3-(hydroxymethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one 1606 (2.79 g,10 mmol) in CH₂Cl₂ (200 mL), MnO₂ (5 g) was added and the resultingmixture was stirred at reflux for 3 h. The mixture was allowed to coolto RT, and concentrated in vacuo. The residue was purified by flashcolumn chromatography on silica gel (10-50% EA/PE) to afford theproduct, 8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinoline-3-carbaldehyde4101 (2.5 g, 90% yield) as a white solid.

8-Methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinoline-3-carbaldehyde 4101(2.4 g, 8.6 mmol) was dissolved in anhydrous THF (280 mL) and cooled to−78° C. under a nitrogen atmosphere. Methyl MgBr (2M, 5 mL, 10 mmol) wasadded slowly, and the resulting mixture was stirred at −78° C. for 2 h.H₂O (5 mL) was added and then the solution was poured into ice-water(200 mL) and extracted with ethyl acetate (3×50 mL). The combinedorganic layer was washed with brine, dried over Na₂SO₄ and filtered. Thefiltrate was concentrated in vacuo, and the residue product was purifiedby flash column chromatography on silica gel (10-50% EA/PE) to affordthe product, 3-(1-hydroxyethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one4102 (1.8 g, 71% yield) as a white solid.

To a solution of3-(1-hydroxyethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one 4102 (1.6 g,5.5 mmol) in CH₂Cl₂, PPh₃ (2.88 g, 11.0 mmol) was added and theresulting mixture was stirred at RT for 30 min. Then CBr₄ (3.64 g, 11.0mmol) was added in portions to the mixture at 0° C. The resultingmixture was allowed to warm to RT, stirred for 30 min, and concentratedin vacuo. The crude product was purified by flash column chromatographyon silica gel (30-50% EA/PE) to afford the desired product,3-(1-bromoethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one 4103 (1.8 g,91% yield) as a white solid.

To a stirred solution of 9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine4103 (436 mg 2 mmol) in anhydrous DMF (10 mL), NaH (60% in mineral oil,77 mg, 2 mmol) was added and the mixture was stirred for 30 min.3-(1-Bromoethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one 4104 (700 mg, 2mmol) was added. The mixture was stirred for 2 h, poured into ice-water(200 mL) and extracted with ethyl acetate (3×50 mL). The combinedorganic layer was washed with brine (20 mL), dried over Na₂SO₄ andfiltered. The filtrate was concentrated in vacuo and the residue waspurified by flash column chromatography on silica gel (10-50% MeOH/DCM)to afford the product,8-methyl-3-(1-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-ylamino)ethyl)-2-o-tolylisoquinolin-1(2H)-one4105 (500 mg, 51% yield) as a white solid.

8-Methyl-3-(1-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-ylamino)ethyl)-2-o-tolylisoquinolin-1(2H)-one 4105 (180 mg, 0.36 mmol) was dissolved in MeOH (HCl) (50 mL)and stirred for 2 h. Aqueous NaHCO₃ solution was added to the reactionmixture and the pH value was adjusted to 9. The mixture was thenfiltered and the filtrate was concentrated in vacuo to afford thedesired product,3-(1-(9H-purin-6-ylamino)ethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one4106 (80 mg, 54% yield) as a yellow solid.

Example 14: Synthesis of3-(4-amino-1-((8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinolin-3-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-5-fluorophenyldihydrogen phosphate

3-((4-Amino-3-(3-fluoro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one4301 (250 mg, 0.5 mmol) was dissolved in anhydrous THF (15 mL) in around bottom flask in dark (covered by aluminum foil) and cooled to 0°C. under an argon atmosphere. CBr₄ (498 mg, 1.5 mmol) was added followedby diethylphosphite (129 μL, 1.0 mmol) and triethylamine (417 μL, 1.5mmol). The resulting mixture was stirred in dark from 0° C. to RT for 16h. The mixture was then partitioned between ethyl acetate and brine. Theorganic layer was dried over Na₂SO₄, filtered and concentrated in vacuo.The residue was purified by column chromatography on silica gel elutingwith methanol and dichloromethane to afford the desired product,3-(4-amino-1-((8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinolin-3-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-5-fluorophenyldiethyl phosphate 4302 (200 mg, 62% yield) as an off-white solid.

3-(4-Amino-1-((8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinolin-3-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-5-fluorophenyldiethyl phosphate 4302 (170 mg, 0.26 mmol) was dissolved in anhydrousCH₃CN (5 mL) and cooled to 0° C. under an argon atmosphere. TMSBr (0.34mL, 2.64 mmol) was slowly added via a syringe and the resulting mixturewas stirred from 0° C. to RT for 16 h. LC-MS showed small amount ofstarting material left, additional amount of TMSBr (0.1 mL) was addedand stirred at RT for 5 h. LC-MS showed the complete conversion. Themixture was concentrated in vacuo, and the residue was dissolved in Et₂O(10 mL) and H₂O (0.5 mL) and stirred for 30 min. The mixture wasconcentrated in vacuo to affords the desired product,3-(4-amino-1-((8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinolin-3-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-5-fluorophenyldihydrogen phosphate 4903 (140 mg, 91% yield).

Example 15: IC50 Values for Selected Compounds

TABLE 4 In Vitro IC₅₀ data for selected compounds. + (greater than 10 ++(less than 10 +++ (less than 1 ++++ (less than IC50 (nM) microMolar)microMolar) microMolar 100 nM) PI3K δ Compound No. Compound No. CompoundNo. Compound No. 1, 5, 22, 27, 38, 39, 4, 14, 15, 17, 18, 21, 2, 3, 6,7, 8, 9, 10, 40, 41, 46, 92, 117, 26, 29, 31, 32, 34, 11, 12, 13, 16,19, 118, 120, 129, 132, 35, 36, 42, 43, 44, 20, 23, 24, 25, 28, 164,165, 172, 188, 45, 47, 49, 57, 69, 30, 33, 37, 48, 50, 190, 197, 198,205, 71, 85, 87, 94, 106, 51, 52, 53, 54, 55, 208, 210, 212, 214, 107,140, 143, 175, 56, 58, 59, 60, 61, 217, 218, 220, 222, 179, 183, 184,191, 62, 63, 64, 65, 66, 237, 263, 285, 294, 193, 196, 199, 200, 67, 68,70, 72, 73, 298 201, 202, 206, 207, 74, 75, 76, 77, 78, 211, 213, 215,219, 79, 80, 81, 82, 83, 224, 225, 228, 229, 84, 86, 88, 89, 90, 230,232, 233, 239, 91, 93, 95, 96, 97, 241, 243, 245, 253, 98, 99, 100, 101,254, 255, 260, 262, 102, 103, 104, 105, 270, 293, 299 108, 109, 110,111, 112, 113, 114, 115, 119, 123, 124, 125, 126, 128, 134, 135, 136,137, 138, 139, 141, 142, 144, 145, 146, 147, 148, 149, 150, 151. 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 166, 167, 168, 169,170, 171, 173, 174, 176, 177, 178, 180, 189, 192, 194, 195, 203, 204,209, 216, 221, 223, 226, 227, 231, 234, 235, 236, 238, 240, 242, 244,246, 247, 248, 249, 250, 251, 252, 256, 257, 258, 259, 261, 264, 265,266, 267, 268, 269, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,281, 282, 283, 284, 286, 287, 288, 289, 290, 291, 292, 295, 296, 297,300, 301, 302, 303, 304 PI3K γ Compound No. Compound No. Compound No.Compound No. 1, 4, 5, 18, 38, 43, 17, 34, 35, 37, 38, 2, 8, 9, 10, 11,14, 3, 6, 7, 12, 13, 16, 60, 69, 169, 172, 40, 42, 57, 61, 65, 15, 20,22, 27, 28, 19, 21, 23, 24, 25, 196, 197, 198, 200, 91, 92, 94, 105,107, 39, 41, 46, 47, 49, 26, 29, 30, 31, 33, 201, 202, 204, 206, 140,164, 170, 175, 51, 55, 58, 66, 70, 36, 44, 45, 48, 50, 207, 208, 209,210, 179, 184, 188, 190, 71, 73, 76, 78, 80, 52, 53, 54, 56, 59, 211,212, 213, 214, 191, 193, 199, 203, 93, 98, 99, 100, 103, 62, 63, 64, 67,68, 215, 217, 218, 219, 216, 224, 229, 232, 104, 106, 108, 109, 72, 74,75, 77, 79, 220, 221, 222, 223, 235, 241, 243, 244, 161, 162, 163, 165,81, 82, 83, 84, 86, 225, 228, 230, 236, 251, 253, 254, 255, 166, 180,192, 205, 87, 88, 89, 90, 95, 237, 239, 245, 266, 263, 264, 268, 270,226, 227, 231, 233, 96, 97, 101, 102, 285, 293, 294, 295, 272, 276, 282,287 240, 242, 249, 250, 142, 145, 146, 147, 296, 297, 298, 299, 252,256, 257, 259, 148, 149, 150, 151, 300, 301, 302, 303, 262, 265, 273,274, 152, 160, 167, 168, 304 275, 278, 280, 286, 171, 173, 174, 176,288, 291 177, 178. 183, 189, 194, 195, 234, 238, 246, 247, 248, 258,260, 261, 267, 269, 271, 277, 279, 281, 283, 284, 289, 290, 292 PI3K αCompound No. Compound No. Compound No. Compound No. 6, 8, 9, 10, 11, 12,13, 3, 7, 63, 66, 84, 86, 53, 95, 101, 102, 142, 148, 150, 153, 14, 15,16, 17, 18, 89, 90, 97, 108, 113, 145, 147, 149, 151, 154, 155, 156,157, 19, 20, 21, 22, 23, 115, 152, 168, 171, 177 158, 159, 176 24, 25,26, 27, 28, 173, 189, 194, 296 29, 30, 31, 32, 33, 34, 35, 36, 37, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 54, 55, 56, 57, 58,59, 60, 61, 62, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 79, 80, 81, 82,83, 85, 87, 88, 91, 93, 96, 98, 99, 100, 103, 104, 105, 106, 107, 109,110, 111, 112, 114, 140, 146, 160, 161, 162, 163, 164, 165, 166, 167,169, 170, 172, 174, 175, 179, 180, 183, 184, 188, 190, 191, 192, 193,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250,251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278,279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292,293, 294, 295, 297, 298, 299, 300, 301, 302, 303, 304 PI3K β CompoundNo. Compound No. Compound No. Compound No. 8, 9, 10, 11, 14, 21, 3, 12,13, 23, 25, 53, 7, 62, 66, 82, 89, 90, 101, 142, 155, 156, 22, 24, 26,27, 28, 55, 58, 61, 63, 65, 95, 97, 100, 102, 157 29, 34, 35, 36, 37,67, 71, 72, 74, 75, 150, 153, 159, 176, 38, 39, 40, 41, 42, 77, 81, 82,83, 84, 189, 289, 292, 296 43, 44, 46, 52, 54, 85, 86, 96, 99, 106, 56,57, 59, 60, 64, 108, 110, 111, 113, 68, 69, 70, 73, 76, 114, 115, 145,147, 78, 79, 80, 87, 88, 149, 151, 154, 158, 91, 93, 98, 103, 104, 160,161, 167, 168, 105, 107, 109, 112, 171, 173, 174, 177, 140, 146, 152,162, 178, 194, 195, 267, 163, 164, 165, 166, 269, 271, 275, 277, 169,170, 172, 175, 278, 279, 281, 282, 179, 180, 183, 184, 283, 284, 286,288, 188, 190, 191, 192, 290, 291, 295, 297 193, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 268, 270, 272,273, 274, 276, 280, 285, 287, 293, 294, 298, 299, 300, 301, 302, 303,304 B cell proliferation Compound No. Compound No. Compound No. CompoundNo. EC₅₀ (nM) 38, 162, 205, 228, 1, 2, 5, 22, 26, 27, 4, 8, 9, 10, 11,14, 3, 6, 7, 12, 13, 16, 229 39, 40, 43, 49, 57, 15, 18, 19, 20, 21, 17,23, 33, 37, 44, 71, 87, 112, 200, 24, 25, 28, 29, 30, 48, 53, 54, 55,62, 201, 202, 208, 209, 31, 32, 34, 35, 36, 63, 66, 67, 68, 72, 210,211, 212, 213, 41, 42, 45, 46, 47, 73, 74, 75, 81, 82, 214, 215, 217,218, 50, 51, 61, 69, 70, 83, 84, 88, 89, 90, 219, 220, 222, 223, 76, 77,78, 79, 80, 93, 95, 96, 97, 99, 232, 233, 237, 262, 85, 86, 91, 98, 100,101, 102, 108, 109, 263, 293 103, 104, 105, 106, 113, 115, 123, 125,107, 110, 111, 114, 126, 128, 134, 136, 119, 124, 133, 135, 137, 138,139, 141, 145, 152, 161, 162, 142, 144, 146, 147, 163, 169, 203, 204,148, 149, 150, 151, 206, 207, 216, 221, 153, 154, 155, 156, 224, 225,226, 230, 157, 158, 159, 160, 243, 245, 246, 251, 166, 167, 168, 170,252, 253, 254, 256, 171, 173, 174, 176, 257, 260, 261, 264, 177, 178,180, 189, 265, 272, 276, 287, 192, 194, 195, 199, 288 227, 231, 234,235, 236, 238, 242, 244, 247, 248, 249, 250, 255, 258, 259, 266, 268,271, 275, 277, 278, 279, 280, 281, 282, 283, 284, 286, 289, 290, 291,292, 295, 296, 297

TABLE 5 Structures of the Compounds for the IC50 results described inTable 4. Structure

Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

Compound 7

Compound 8

Compound 9

Compound 10

Compound 11

Compound 12

Compound 13

Compound 14

Compound 15

Compound 16

Compound 17

Compound 18

Compound 19

Compound 20

Compound 21

Compound 22

Compound 23

Compound 24

Compound 25

Compound 26

Compound 27

Compound 28

Compound 29

Compound 30

Compound 31

Compound 32

Compound 33

Compound 34

Compound 35

Compound 36

Compound 37

Compound 38

Compound 39

Compound 40

Compound 41

Compound 42

Compound 43

Compound 44

Compound 45

Compound 46

Compound 47

Compound 48

Compound 49

Compound 50

Compound 51

Compound 52

Compound 53

Compound 54

Compound 55

Compound 56

Compound 57

Compound 58

Compound 59

Compound 60

Compound 61

Compound 62

Compound 63

Compound 64

Compound 65

Compound 66

Compound 67

Compound 68

Compound 69

Compound 70

Compound 71

Compound 72

Compound 73

Compound 74

Compound 75

Compound 76

Compound 77

Compound 78

Compound 79

Compound 80

Compound 81

Compound 82

Compound 83

Compound 84

Compound 85

Compound 86

Compound 87

Compound 88

Compound 89

Compound 90

Compound 91

Compound 92

Compound 93

Compound 94

Compound 95

Compound 96

Compound 97

Compound 98

Compound 99

Compound 100

Compound 101

Compound 102

Compound 103

Compound 104

Compound 105

Compound 106

Compound 107

Compound 108

Compound 109

Compound 110

Compound 111

Compound 112

Compound 113

Compound 114

Compound 115

Compound 116

Compound 117

Compound 118

Compound 119

Compound 120

Compound 121

Compound 122

Compound 123

Compound 124

Compound 125

Compound 126

Compound 127

Compound 128

Compound 129

Compound 130

Compound 131

Compound 132

Compound 133

Compound 134

Compound 135

Compound 136

Compound 137

Compound 138

Compound 139

Compound 140

Compound 141

Compound 142

Compound 143

Compound 144

Compound 145

Compound 146

Compound 147

Compound 148

Compound 149

Compound 150

Compound 151

Compound 152

Compound 153

Compound 154

Compound 155

Compound 156

Compound 157

Compound 158

Compound 159

Compound 160

Compound 161

Compound 162

Compound 163

Compound 164

Compound 165

Compound 166

Compound 167

Compound 168

Compound 169

Compound 170

Compound 171

Compound 172

Compound 173

Compound 174

Compound 175

Compound 176

Compound 177

Compound 178

Compound 179

Compound 180

Compound 183

Compound 184

Compound 188

Compound 189

Compound 190

Compound 191

Compound 192

Compound 193

Compound 194

Compound 195

Compound 196

Compound 197

Compound 198

Compound 199

Compound 200

Compound 201

Compound 202

Compound 203

Compound 204

Compound 205

Compound 206

Compound 207

Compound 208

Compound 209

Compound 210

Compound 211

Compound 212

Compound 213

Compound 214

Compound 215

Compound 216

Compound 217

Compound 218

Compound 219

Compound 220

Compound 221

Compound 222

Compound 223

Compound 224

Compound 225

Compound 226

Compound 227

Compound 228

Compound 229

Compound 230

Compound 231

Compound 232

Compound 233

Compound 234

Compound 235

Compound 236

Compound 237

Compound 238

Compound 239

Compound 240

Compound 241

Compound 242

Compound 243

Compound 244

Compound 245

Compound 246

Compound 247

Compound 248

Compound 249

Compound 250

Compound 251

Compound 252

Compound 253

Compound 254

Compound 255

Compound 256

Compound 257

Compound 258

Compound 259

Compound 260

Compound 261

Compound 262

Compound 263

Compound 264

Compound 265

Compound 266

Compound 267

Compound 268

Compound 269

Compound 270

Compound 271

Compound 272

Compound 273

Compound 274

Compound 275

Compound 276

Compound 277

Compound 278

Compound 279

Compound 280

Compound 281

Compound 282

Compound 283

Compound 284

Compound 285

Compound 286

Compound 287

Compound 288

Compound 289

Compound 290

Compound 291

Compound 292

Compound 293

Compound 294

Compound 295

Compound 296

Compound 297

Compound 298

Compound 299

Compound 300

Compound 301

Compound 302

Compound 303

Compound 304

Example 16: Expression and Inhibition Assays of p110α/p85α, p110β/p85α,p110δ/p85α, and p110γ

Class I PI3-Ks can be either purchased (p110α/p85α, p110β/p85α,p110δ/p85α from Upstate, and p110γ from Sigma) or expressed aspreviously described (Knight et al., 2004). IC50 values are measuredusing either a standard TLC assay for lipid kinase activity (describedbelow) or a high-throughput membrane capture assay. Kinase reactions areperformed by preparing a reaction mixture containing kinase, inhibitor(2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mMMgCl2), and freshly sonicated phosphatidylinositol (100 μg/ml).Reactions are initiated by the addition of ATP containing 10 μCi ofγ-32P-ATP to a final concentration 10 or 100 μM and allowed to proceedfor 5 minutes at room temperature. For TLC analysis, reactions are thenterminated by the addition of 105 μl 1N HCl followed by 160 μlCHCl3:MeOH (1:1). The biphasic mixture is vortexed, briefly centrifuged,and the organic phase is transferred to a new tube using a gel loadingpipette tip precoated with CHCl₃. This extract is spotted on TLC platesand developed for 3-4 hours in a 65:35 solution of n-propanol:1M aceticacid. The TLC plates are then dried, exposed to a phosphorimager screen(Storm, Amersham), and quantitated. For each compound, kinase activityis measured at 10-12 inhibitor concentrations representing two-folddilutions from the highest concentration tested (typically, 200 μM). Forcompounds showing significant activity, IC50 determinations are repeatedtwo to four times, and the reported value is the average of theseindependent measurements.

Other commercial kits or systems for assaying PI3-K activities areavailable. The commercially available kits or systems can be used toscreen for inhibitors and/or agonists of PI3-Ks including but notlimited to PI 3-Kinase α, β, δ, and γ. An exemplary system is PI3-Kinase (human) HTRF™ Assay from Upstate. The assay can be carried outaccording to the procedures suggested by the manufacturer. Briefly, theassay is a time resolved FRET assay that indirectly measures PIP3product formed by the activity of a PI3-K. The kinase reaction isperformed in a microtiter plate (e.g., a 384 well microtiter plate). Thetotal reaction volume is approximately 20 ul per well. In the firststep, each well receives 2 ul of test compound in 20% dimethylsulphoxideresulting in a 2% DMSO final concentration. Next, approximately 14.5 ulof a kinase/PIP2 mixture (diluted in 1× reaction buffer) is added perwell for a final concentration of 0.25-0.3 ug/ml kinase and 10 uM PIP2.The plate is sealed and incubated for 15 minutes at room temperature. Tostart the reaction, 3.5 ul of ATP (diluted in 1× reaction buffer) isadded per well for a final concentration of 10 uM ATP. The plate issealed and incubated for 1 hour at room temperature. The reaction isstopped by adding 5 ul of Stop Solution per well and then 5 ul ofDetection Mix is added per well. The plate is sealed, incubated for 1hour at room temperature, and then read on an appropriate plate reader.Data is analyzed and IC50s are generated using GraphPad Prism 5.

Example 17: Expression and Inhibition Assays of Abl

The cross-activity or lack thereof of one or more compounds of thepresent invention against Abl kinase can be measured according to anyprocedures known in the art or methods disclosed below. The compoundsdescribed herein can be assayed in triplicate against recombinantfull-length Abl or Abl (T315I) (Upstate) in an assay containing 25 mMHEPES, pH 7.4, 10 mM MgCl2, 200 μM ATP (2.5 μCi of γ-32P-ATP), and 0.5mg/mL BSA. The optimized Abl peptide substrate EAIYAAPFAKKK (SEQ IDNO: 1) is used as phosphoacceptor (200 μM). Reactions are terminated byspotting onto phosphocellulose sheets, which are washed with 0.5%phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets aredried and the transferred radioactivity quantitated by phosphorimaging.

Example 18: Expression and Inhibition Assays of Hck

The cross-activity or lack thereof of one or more compounds of thepresent invention against Hck kinase can be measured according to anyprocedures known in the art or methods disclosed below. The compoundsdescribed herein can be assayed in triplicate against recombinantfull-length Hck in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl2,200 μM ATP (2.5 μCi of γ-32P-ATP), and 0.5 mg/mL BSA. The optimized Srcfamily kinase peptide substrate EIYGEFKKK (SEQ ID NO: 2) is used asphosphoacceptor (200 μM). Reactions are terminated by spotting ontophosphocellulose sheets, which are washed with 0.5% phosphoric acid(approximately 6 times, 5-10 minutes each). Sheets are dried and thetransferred radioactivity quantitated by phosphorimaging.

Example 19: Expression and Inhibition Assays of Inulsin Receptor (IR)

The cross-activity or lack thereof of one or more compounds of thepresent invention against IR receptor kinase can be measured accordingto any procedures known in the art or methods disclosed below. Thecompounds described herein can be assayed in triplicate againstrecombinant insulin receptor kinase domain (Upstate) in an assaycontaining 25 mM HEPES, pH 7.4, 10 mM MgCl2, 10 mM MnCl2, 200 μM ATP(2.5 μCi of γ-32P-ATP), and 0.5 mg/mL BSA. Poly E-Y (Sigma; 2 mg/mL) isused as a substrate. Reactions are terminated by spotting ontonitrocellulose, which is washed with 1M NaCl/1% phosphoric acid(approximately 6 times, 5-10 minutes each). Sheets are dried and thetransferred radioactivity quantitated by phosphorimaging.

Example 20: Expression and Inhibition Assays of Src

The cross-activity or lack thereof of one or more compounds of thepresent invention against Src kinase can be measured according to anyprocedures known in the art or methods disclosed below. The compoundsdescribed herein can be assayed in triplicate against recombinantfull-length Src or Src (T338I) in an assay containing 25 mM HEPES, pH7.4, 10 mM MgCl2, 200 μM ATP (2.5 μCi of γ-32P-ATP), and 0.5 mg/mL BSA.The optimized Src family kinase peptide substrate EIYGEFKKK (SEQ ID NO:2) is used as phosphoacceptor (200 μM). Reactions are terminated byspotting onto phosphocellulose sheets, which are washed with 0.5%phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets weredried and the transferred radioactivity quantitated by phosphorimaging.

Example 21: Expression and Inhibition Assays of DNA-PK (DNAK)

The cross-activity or lack thereof of one or more compounds of thepresent invention against DNAK kinase can be measured according to anyprocedures known in the art. DNA-PK can be purchased from Promega andassayed using the DNA-PK Assay System (Promega) according to themanufacturer's instructions.

Example 22: Expression and Inhibition Assays mTOR

The cross-activity or lack thereof of one or more compounds of thepresent invention against mTor can be measured according to anyprocedures known in the art or methods disclosed below. The compoundsdescribed herein can be tested against recombinant mTOR (Invitrogen) inan assay containing 50 mM HEPES, pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2.5 mM,0.01% Tween, 10 μM ATP (2.5 μCi of μ-32P-ATP), and 3 μg/mL BSA. Ratrecombinant PHAS-1/4EBP1 (Calbiochem; 2 mg/mL) is used as a substrate.Reactions are terminated by spotting onto nitrocellulose, which iswashed with 1M NaCl/1% phosphoric acid (approximately 6 times, 5-10minutes each). Sheets are dried and the transferred radioactivityquantitated by phosphorimaging.

Other kits or systems for assaying mTOR activity are commerciallyavailable. For instance, one can use Invitrogen's LanthaScreen™ Kinaseassay to test the inhibitors of mTOR disclosed herein. This assay is atime resolved FRET platform that measures the phosphorylation of GFPlabeled 4EBP1 by mTOR kinase. The kinase reaction is performed in awhite 384 well microtiter plate. The total reaction volume is 20 ul perwell and the reaction buffer composition is 50 mM HEPES pH7.5, 0.01%Polysorbate 20, 1 mM EGTA, 10 mM MnCl2, and 2 mM DTT. In the first step,each well receives 2 ul of test compound in 20% dimethylsulphoxideresulting in a 2% DMSO final concentration. Next, 8 ul of mTOR dilutedin reaction buffer is added per well for a 60 ng/ml final concentration.To start the reaction, 10 ul of an ATP/GFP-4EBP1 mixture (diluted inreaction buffer) is added per well for a final concentration of 10 uMATP and 0.5 uM GFP-4EBP1. The plate is sealed and incubated for 1 hourat room temperature. The reaction is stopped by adding 10 ul per well ofa Tb-anti-pT46 4EBP1 antibody/EDTA mixture (diluted in TR-FRET buffer)for a final concentration of 1.3 nM antibody and 6.7 mM EDTA. The plateis sealed, incubated for 1 hour at room temperature, and then read on aplate reader set up for LanthaScreen™ TR-FRET. Data is analyzed andIC50s are generated using GraphPad Prism 5.

Example 23: Expression and Inhibition Assays of Vascular EndothelialGrowth Receptor

The cross-activity or lack thereof of one or more compounds of thepresent invention against VEGF receptor can be measured according to anyprocedures known in the art or methods disclosed below. The compoundsdescribed herein can be tested against recombinant KDR receptor kinasedomain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mMMgCl2, 0.1% BME, 10 μM ATP (2.5 μCi of μ-32P-ATP), and 3 μg/mL BSA. PolyE-Y (Sigma; 2 mg/mL) is used as a substrate. Reactions are terminated byspotting onto nitrocellulose, which is washed with 1M NaCl/1% phosphoricacid (approximately 6 times, 5-10 minutes each). Sheets are dried andthe transferred radioactivity quantitated by phosphorimaging.

Example 24: Expression and Inhibition Assays of Ephrin Receptor B4(EphB4)

The cross-activity or lack thereof of one or more compounds of thepresent invention against EphB4 can be measured according to anyprocedures known in the art or methods disclosed below. The compoundsdescribed herein can be tested against recombinant Ephrin receptor B4kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4,10 mM MgCl2, 0.1% BME, 10 μM ATP (2.5 μCi of μ-32P-ATP), and 3 μg/mLBSA. Poly E-Y (Sigma; 2 mg/mL) is used as a substrate. Reactions areterminated by spotting onto nitrocellulose, which is washed with 1MNaCl/1% phosphoric acid (approximately 6 times, 5-10 minutes each).Sheets are dried and the transferred radioactivity quantitated byphosphorimaging.

Example 25: Expression and Inhibition Assays of Epidermal Growth FactorReceptor (EGFR)

The cross-activity or lack thereof of one or more compounds of thepresent invention against EGFR kinase can be measured according to anyprocedures known in the art or methods disclosed below. The compoundsdescribed herein can be tested against recombinant EGF receptor kinasedomain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mMMgCl2, 0.1% BME, 10 μM ATP (2.5 μCi of μ-32P-ATP), and 3 μg/mL BSA. PolyE-Y (Sigma; 2 mg/mL) is used as a substrate. Reactions are terminated byspotting onto nitrocellulose, which is washed with 1M NaCl/1% phosphoricacid (approximately 6 times, 5-10 minutes each). Sheets are dried andthe transferred radioactivity quantitated by phosphorimaging.

Example 26: Expression and Inhibition Assays of KIT Assay

The cross-activity or lack thereof of one or more compounds of thepresent invention against KIT kinase can be measured according to anyprocedures known in the art or methods disclosed below. The compoundsdescribed herein can be tested against recombinant KIT kinase domain(Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl2, 1mM DTT, 10 mM MnCl2, 10 μM ATP (2.5 μCi of μ-32P-ATP), and 3 μg/mL BSA.Poly E-Y (Sigma; 2 mg/mL) is used as a substrate. Reactions areterminated by spotting onto nitrocellulose, which is washed with 1MNaCl/1% phosphoric acid (approximately 6 times, 5-10 minutes each).Sheets are dried and the transferred radioactivity quantitated byphosphorimaging.

Example 27: Expression and Inhibition Assays of RET

The cross-activity or lack thereof of one or more compounds of thepresent invention against RET kinase can be measured according to anyprocedures known in the art or methods disclosed below. The compoundsdescribed herein can be tested against recombinant RET kinase domain(Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl2,2.5 mM DTT, 10 μM ATP (2.5 μCi of μ-32P-ATP), and 3 μg/mL BSA. Theoptimized Abl peptide substrate EAIYAAPFAKKK (SEQ ID NO: 1) is used asphosphoacceptor (200 μM). Reactions are terminated by spotting ontophosphocellulose sheets, which are washed with 0.5% phosphoric acid(approximately 6 times, 5-10 minutes each). Sheets are dried and thetransferred radioactivity quantitated by phosphorimaging.

Example 28: Expression and Inhibition Assays of Platelet Derived GrowthFactor Receptor (PDGFR)

The cross-activity or lack thereof of one or more compounds of thepresent invention against PDGFR kinase can be measured according to anyprocedures known in the art or methods disclosed below. The compoundsdescribed herein can be tested against recombinant PDG receptor kinasedomain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mMMgCl2, 2.5 mM DTT, 10 μM ATP (2.5 μCi of μ-32P-ATP), and 3 μg/mL BSA.The optimized Abl peptide substrate EAIYAAPFAKKK (SEQ ID NO: 1) is usedas phosphoacceptor (200 μM). Reactions are terminated by spotting ontophosphocellulose sheets, which are washed with 0.5% phosphoric acid(approximately 6 times, 5-10 minutes each). Sheets are dried and thetransferred radioactivity quantitated by phosphorimaging.

Example 29: Expression and Inhibition Assays of FMS-Related TyrosineKinase 3 (FLT-3)

The cross-activity or lack thereof of one or more compounds of thepresent invention against FLT-3 kinase can be measured according to anyprocedures known in the art or methods disclosed below. The compoundsdescribed herein can be tested against recombinant FLT-3 kinase domain(Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl2,2.5 mM DTT, 10 μM ATP (2.5 μCi of μ-32P-ATP), and 3 μg/mL BSA. Theoptimized Abl peptide substrate EAIYAAPFAKKK (SEQ ID NO: 1) is used asphosphoacceptor (200 μM). Reactions are terminated by spotting ontophosphocellulose sheets, which are washed with 0.5% phosphoric acid(approximately 6 times, 5-10 minutes each). Sheets are dried and thetransferred radioactivity quantitated by phosphorimaging.

Example 30: Expression and Inhibition Assays of TEK Receptor TyrosineKinase (TIE2)

The cross-activity or lack thereof of one or more compounds of thepresent invention against TIE2 kinase can be measured according to anyprocedures known in the art or methods disclosed below. The compoundsdescribed herein can be tested against recombinant TIE2 kinase domain(Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl2, 2mM DTT, 10 mM MnCl2, 10 μM ATP (2.5 μCi of μ-32P-ATP), and 3 μg/mL BSA.Poly E-Y (Sigma; 2 mg/mL) is used as a substrate. Reactions areterminated by spotting onto nitrocellulose, which is washed with 1MNaCl/1% phosphoric acid (approximately 6 times, 5-10 minutes each).Sheets are dried and the transferred radioactivity quantitated byphosphorimaging.

Example 31: B Cell Activation and Proliferation Assay

The ability of one or more subject compounds to inhibit B cellactivation and proliferation is determined according to standardprocedures known in the art. For example, an in vitro cellularproliferation assay is established that measures the metabolic activityof live cells. The assay is performed in a 96 well microtiter plateusing Alamar Blue reduction. Balb/c splenic B cells are purified over aFicoll-Paque™ PLUS gradient followed by magnetic cell separation using aMACS B cell Isolation Kit (Miletenyi). Cells are plated in 90 ul at50,000 cells/well in B Cell Media (RPMI+10% FBS+Penn/Strep+50 uM bME+5mM HEPES). A compound disclosed herein is diluted in B Cell Media andadded in a 10 ul volume. Plates are incubated for 30 min at 37 C and 5%CO₂ (0.2% DMSO final concentration). A 50 ul B cell stimulation cocktailis then added containing either 10 ug/ml LPS or 5 ug/ml F(ab′)2 Donkeyanti-mouse IgM plus 2 ng/ml recombinant mouse IL4 in B Cell Media.Plates are incubated for 72 hours at 37° C. and 5% CO₂. A volume of 15uL of Alamar Blue reagent is added to each well and plates are incubatedfor 5 hours at 37 C and 5% CO₂. Alamar Blue fluoresce is read at560Ex/590Em, and IC50 or EC50 values are calculated using GraphPad Prism5.

Example 32: Tumor Cell Line Proliferation Assay

The ability of one or more subject compounds to inhibit tumor cell lineproliferation is determined according to standard procedures known inthe art. For instance, an in vitro cellular proliferation assay can beperformed to measure the metabolic activity of live cells. The assay isperformed in a 96 well microtiter plate using Alamar Blue reduction.Human tumor cell lines are obtained from ATCC (e.g., MCF7, U-87 MG,MDA-MB-468, PC-3), grown to confluency in T75 flasks, trypsinized with0.25% trypsin, washed one time with Tumor Cell Media (DMEM+10% FBS), andplated in 90 ul at 5,000 cells/well in Tumor Cell Media. A compounddisclosed herein is diluted in Tumor Cell Media and added in a 10 ulvolume. Plates are incubated for 72 hours at 37 C and 5% CO₂. A volumeof 10 uL of Alamar Blue reagent is added to each well and plates areincubated for 3 hours at 37 C and 5% CO₂. Alamar Blue fluoresce is readat 560Ex/590Em, and IC50 values are calculated using GraphPad Prism 5.

Example 33: Antitumor Activity in Vivo

The compounds described herein can be evaluated in a panel of human andmurine tumor models.

Paclitaxel-Refractory Tumor Models

1. Clinically-Derived Ovarian Carcinoma Model.

This tumor model is established from a tumor biopsy of an ovarian cancerpatient. Tumor biopsy is taken from the patient.

The compounds described herein are administered to nude mice bearingstaged tumors using an every 2 days×5 schedule.

2. A2780Tax Human Ovarian Carcinoma Xenograft (Mutated Tubulin).

A2780Tax is a paclitaxel-resistant human ovarian carcinoma model. It isderived from the sensitive parent A2780 line by co-incubation of cellswith paclitaxel and verapamil, an MDR-reversal agent. Its resistancemechanism has been shown to be non-MDR related and is attributed to amutation in the gene encoding the beta-tubulin protein.

The compounds described herein can be administered to mice bearingstaged tumors on an every 2 days×5 schedule.

3. HCT116/VM46 Human Colon Carcinoma Xenograft (Multi-Drug Resistant).

HCT116/VM46 is an MDR-resistant colon carcinoma developed from thesensitive HCT116 parent line. In vivo, grown in nude mice, HCT116/VM46has consistently demonstrated high resistance to paclitaxel.

The compounds described herein can be administered to mice bearingstaged tumors on an every 2 days×5 schedule.

5. M5076 Murine Sarcoma Model

M5076 is a mouse fibrosarcoma that is inherently refractory topaclitaxel in vivo.

The compounds described herein can be administered to mice bearingstaged tumors on an every 2 days×5 schedule.

One or more compounds of the invention can be used in combination othertherapeutic agents in vivo in the multidrug resistant human coloncarcinoma xenografts HCT/VM46 or any other model known in the artincluding those described herein.

Example 34: Microsome Stability Assay

The stability of one or more subject compounds is determined accordingto standard procedures known in the art. For example, stability of oneor more subject compounds is established by an in vitro assay. Inparticular, an in vitro microsome stability assay is established thatmeasures stability of one or more subject compounds when reacting withmouse, rat or human microsomes from liver. The microsome reaction withcompounds is performed in 1.5 mL Eppendorf tube. Each tube contains 0.1μL of 10.0 mg/ml NADPH; 75 μL of 20.0 mg/ml mouse, rat or human livermicrosome; 0.4 μL of 0.2M phosphate buffer, and 425 μL of ddH₂O.Negative control (without NADPH) tube contains 75 μL of 20.0 mg/mlmouse, rat or human liver microsome; 0.4 μL of 0.2M phosphate buffer,and 525 μL of ddH₂O. The reaction is started by adding 1.0 μL of 10.0 mMtested compound. The reaction tubes are incubated at 37° C. 100 μLsample is collected into new Eppendorf tube containing 300 μL coldMethanol at 0, 5, 10, 15, 30 and 60 minutes of reaction. Samples arecentrifuged at 15,000 rpm to remove protein. Supernatant of centrifugedsample is transferred to new tube. Concentration of stable compoundafter reaction with microsome in the supernatant is measured by LiquidChromatography/Mass Spectrometry (LC-MS).

Example 35: Plasma Stability Assay

The stability of one or more subject compounds in plasma is determinedaccording to standard procedures known in the art. See, e.g., RapidCommun. Mass Spectrom., 10: 1019-1026. The following procedure is anHPLC-MS/MS assay using human plasma; other species including monkey,dog, rat, and mouse are also available. Frozen, heparinized human plasmais thawed in a cold water bath and spun for 10 minutes at 2000 rpm at 4°C. prior to use. A subject compound is added from a 400 μM stocksolution to an aliquot of pre-warmed plasma to give a final assay volumeof 400 μL (or 800 μL for half-life determination), containing 5 μM testcompound and 0.5% DMSO. Reactions are incubated, with shaking, for 0minutes and 60 minutes at 37° C., or for 0, 15, 30, 45 and 60 minutes at37 C for half life determination. Reactions are stopped by transferring50 μL of the incubation mixture to 200 μL of ice-cold acetonitrile andmixed by shaking for 5 minutes. The samples are centrifuged at 6000×gfor 15 minutes at 4° C. and 120 μL of supernatant removed into cleantubes. The samples are then evaporated to dryness and submitted foranalysis by HPLC-MS/MS.

Where desired, one or more control or reference compounds (5 μM) aretested simultaneously with the test compounds: one compound,propoxycaine, with low plasma stability and another compound,propantheline, with intermediate plasma stability.

Samples are reconstituted in acetonitrile/methanol/water (1/1/2, v/v/v)and analyzed via (RP)HPLC-MS/MS using selected reaction monitoring(SRM). The HPLC conditions consist of a binary LC pump with autosampler,a mixed-mode, C12, 2×20 mm column, and a gradient program. Peak areascorresponding to the analytes are recorded by HPLC-MS/MS. The ratio ofthe parent compound remaining after 60 minutes relative to the amountremaining at time zero, expressed as percent, is reported as plasmastability. In case of half-life determination, the half-life isestimated from the slope of the initial linear range of the logarithmiccurve of compound remaining (%) vs. time, assuming first order kinetics.

Example 36: Chemical Stability

The chemical stability of one or more subject compounds is determinedaccording to standard procedures known in the art. The following detailsan exemplary procedure for ascertaining chemical stability of a subjectcompound. The default buffer used for the chemical stability assay isphosphate-buffered saline (PBS) at pH 7.4; other suitable buffers can beused. A subject compound is added from a 100 μM stock solution to analiquot of PBS (in duplicate) to give a final assay volume of 400 μL,containing 5 μM test compound and 1% DMSO (for half-life determination atotal sample volume of 700 μL is prepared). Reactions are incubated,with shaking, for 0 minutes and 24 hours at 37° C.; for half-lifedetermination samples are incubated for 0, 2, 4, 6, and 24 hours.Reactions are stopped by adding immediately 100 μL of the incubationmixture to 100 μL of acetonitrile and vortexing for 5 minutes. Thesamples are then stored at −20° C. until analysis by HPLC-MS/MS. Wheredesired, a control compound or a reference compound such as chlorambucil(5 μM) is tested simultaneously with a subject compound of interest, asthis compound is largely hydrolyzed over the course of 24 hours. Samplesare analyzed via (RP)HPLC-MS/MS using selected reaction monitoring(SRM). The HPLC conditions consist of a binary LC pump with autosampler,a mixed-mode, C12, 2×20 mm column, and a gradient program. Peak areascorresponding to the analytes are recorded by HPLC-MS/MS. The ratio ofthe parent compound remaining after 24 hours relative to the amountremaining at time zero, expressed as percent, is reported as chemicalstability. In case of half-life determination, the half-life isestimated from the slope of the initial linear range of the logarithmiccurve of compound remaining (%) vs. time, assuming first order kinetics.

Example 37: Akt Kinase Assay

Cells comprising components of the Akt/mTOR pathway, including but notlimited to L6 myoblasts, B-ALL cells, B-cells, T-cells, leukemia cells,bone marrow cells, p190 transduced cells, philladelphia chromosomepositive cells (Ph+), and mouse embryonic fibroblasts, are typicallygrown in cell growth media such as DMEM supplemented with fetal bovineserum and/or antibiotics, and grown to confluency.

In order to compare the effect of one or more compounds disclosed hereinon Akt activation, said cells are serum starved overnight and incubatedwith one or more compounds disclosed herein or about 0.1% DMSO forapproximately 1 minute to about 1 hour prior to stimulation with insulin(e.g. 100 nM) for about 1 minutes to about 1 hour. Cells are lysed byscraping into ice cold lysis buffer containing detergents such as sodiumdodecyl sulfate and protease inhibitors (e.g., PMSF). After contactingcells with lysis buffer, the solution is briefly sonicated, cleared bycentrifugation, resolved by SDS-PAGE, transferred to nitrocellulose orPVDF and immunoblotted using antibodies to phospho-Akt S473, phospho-AktT308, Akt, and β-actin (Cell Signaling Technologies).

The results demonstrate that one or more compounds of the presentdisclosure inhibit insulin stimulated phosphorylation of Akt at S473.Alternatively, some compounds disclosed herein additionally inhibitinsulin stimulated phosphorylation of Akt at T308. Such class ofcompounds can inhibit Akt more effectively than rapamycin and may beindicative of mTORC2 inhibitors or inhibitors of upstream kinases suchas PI3K or Akt.

Example 38: Kinase Signaling in Blood

PI3K/Akt/mTor signaling is measured in blood cells using the phosflowmethod (Methods Enzymol. 2007; 434:131-54). The advantage of this methodis that it is by nature a single cell assay so that cellularheterogeneity can be detected rather than population averages. Thisallows concurrent dinstinction of signaling states in differentpopulations defined by other markers. Phosflow is also highlyquantitative. To test the effects of one or more compounds disclosedherein, unfractionated splenocytes, or peripheral blood mononuclearcells are stimulated with anti-CD3 to initiate T-cell receptorsignaling. The cells are then fixed and stained for surface markers andintracellular phosphoproteins. It is expected that inhibitors disclosedherein inhibit anti-CD3 mediated phosphorylation of Akt-S473 and S6,whereas rapamycin inhibits S6 phosphorylation and enhances Aktphosphorylation under the conditions tested.

Similarly, aliquots of whole blood are incubated for 15 minutes withvehicle (e.g. 0.1% DMSO) or kinase inhibitors at various concentrations,before addition of stimuli to crosslink the T cell receptor (TCR)(anti-CD3 with secondary antibody) or the B cell receptor (BCR) usinganti-kappa light chain antibody (Fab′2 fragments). After approximately 5and 15 minutes, samples are fixed (e.g. with cold 4% paraformaldehyde)and used for phosflow. Surface staining is used to distinguish T and Bcells using antibodies directed to cell surface markers that are knownto the art. The level of phosphorylation of kinase substrates such asAkt and S6 are then measured by incubating the fixed cells with labeledantibodies specific to the phosphorylated isoforms of these proteins.The population of cells are then analyzed by flow cytometry.

Example 39: Colony Formation Assay

Murine bone marrow cells freshly transformed with a p190 BCR-Ablretrovirus (herein referred to as p190 transduced cells) are plated inthe presence of various drug combinations in M3630 methylcellulose mediafor about 7 days with recombinant human IL-7 in about 30% serum, and thenumber of colonies formed is counted by visual examination under amicroscope.

Alternatively, human peripheral blood mononuclear cells are obtainedfrom Philadelphia chromosome positive (Ph+) and negative (Ph−) patientsupon initial diagnosis or relapse. Live cells are isolated and enrichedfor CD19+CD34+ B cell progenitors. After overnight liquid culture, cellsare plated in methocult GF+H4435, Stem Cell Tehcnologies) supplementedwith cytokines (IL-3, IL-6, IL-7, G-CSF, GM-CSF, CF, Flt3 ligand, anderythropoietin) and various concentrations of known chemotherapeuticagents in combination with either compounds of the present disclosure.Colonies are counted by microscopy 12-14 days later. This method can beused to test for evidence of additive or synergistic activity.

Example 40: In Vivo Effect of Kinase Inhibitors on Leukemic Cells

Female recipient mice are lethally irradiated from a γ source in twodoses about 4 hr apart, with approximately 5Gy each. About 1 hr afterthe second radiation dose, mice are injected i.v. with about 1×10⁶leukemic cells (e.g. Ph+ human or murine cells, or p190 transduced bonemarrow cells). These cells are administered together with aradioprotective dose of about 5×10⁶ normal bone marrow cells from 3-5week old donor mice. Recipients are given antibiotics in the water andmonitored daily. Mice who become sick after about 14 days are euthanizedand lymphoid organs are harvested for analysis. Kinase inhibitortreatment begins about 10 days after leukemic cell injection andcontinues daily until the mice become sick or a maximum of approximately35 days post-transplant. Inhibitors are given by oral lavage.

Peripheral blood cells are collected approximately on day 10(pre-treatment) and upon euthanization (post treatment), contacted withlabeled anti-hCD4 antibodies and counted by flow cytometry. This methodcan be used to demonstrate that the synergistic effect of one or morecompounds disclosed herein in combination with known chemotherapeuticagents significantly reduce leukemic blood cell counts as compared totreatment with known chemotherapeutic agents (e.g. Gleevec) alone underthe conditions tested.

Example 41: Treatment of Lupus Disease Model Mice

Mice lacking the inhibitory receptor FcγRIIb that opposes PI3K signalingin B cells develop lupus with high penetrance. FcγRIIb knockout mice(R2KO, Jackson Labs) are considered a valid model of the human diseaseas some lupus patients show decreased expression or function of FcγRIIb(S. Bolland and J. V. Ravtech 2000. Immunity 12:277-285).

The R2KO mice develop lupus-like disease with anti-nuclear antibodies,glomerulonephritis and proteinurea within about 4-6 months of age. Forthese experiments, the rapamycin analogue RAD001 (available from LCLaboratories) is used as a benchmark compound, and administered orally.This compound has been shown to ameliorate lupus symptoms in theB6.Sle1z.Sle3z model (T. Wu et al. J. Clin Invest. 117:2186-2196).

Lupus disease model mice such as R2KO, BXSB or MLR/lpr are treated atabout 2 months old, approximately for about two months. Mice are givendoses of: vehicle, RAD001 at about 10 mg/kg, or compounds disclosedherein at approximately 1 mg/kg to about 500 mg/kg. Blood and urinesamples are obtained at approximately throughout the testing period, andtested for antinuclear antibodies (in dilutions of serum) or proteinconcentration (in urine). Serum is also tested for anti-ssDNA andanti-dsDNA antibodies by ELISA. Animals are euthanized at day 60 andtissues harvested for measuring spleen weight and kidney disease.Glomerulonephritis is assessed in kidney sections stained with H&E.Other animals are studied for about two months after cessation oftreatment, using the same endpoints.

This model established in the art can be employed to demonstrate thatthe kinase inhibitors disclosed herein can suppress or delay the onsetof lupus symptoms in lupus disease model mice.

Example 42: Murine Bone Marrow Transplant Assay

Female recipient mice are lethally irradiated from a γ ray source. About1 hr after the radiation dose, mice are injected with about 1×106leukemic cells from early passage p190 transduced cultures (e.g. asdescribed in Cancer Genet Cytogenet. 2005 August; 161(1):51-6). Thesecells are administered together with a radioprotective dose ofapproximately 5×106 normal bone marrow cells from 3-5 wk old donor mice.Recipients are given antibiotics in the water and monitored daily. Micewho become sick after about 14 days are euthanized and lymphoid organsharvested for flow cytometry and/or magnetic enrichment. Treatmentbegins on approximately day 10 and continues daily until mice becomesick, or after a maximum of about 35 days post-transplant. Drugs aregiven by oral gavage (p.o.). In a pilot experiment a dose ofchemotherapeutic that is not curative but delays leukemia onset by aboutone week or less is identified; controls are vehicle-treated or treatedwith chemotherapeutic agent, previously shown to delay but not cureleukemogenesis in this model (e.g. imatinib at about 70 mg/kg twicedaily). For the first phase p190 cells that express eGFP are used, andpostmortem analysis is limited to enumeration of the percentage ofleukemic cells in bone marrow, spleen and lymph node (LN) by flowcytometry. In the second phase, p190 cells that express a tailless formof human CD4 are used and the postmortem analysis includes magneticsorting of hCD4+ cells from spleen followed by immunoblot analysis ofkey signaling endpoints: p Akt-T308 and S473; pS6 and p4EBP-1. Ascontrols for immunoblot detection, sorted cells are incubated in thepresence or absence of kinase inhibitors of the present disclosureinhibitors before lysis. Optionally, “phosflow” is used to detect pAkt-S473 and pS6-S235/236 in hCD4-gated cells without prior sorting.These signaling studies are particularly useful if, for example,drug-treated mice have not developed clinical leukemia at the 35 daytime point. Kaplan-Meier plots of survival are generated and statisticalanalysis done according to methods known in the art. Results from p190cells are analyzed separated as well as cumulatively.

Samples of peripheral blood (100-200 μl) are obtained weekly from allmice, starting on day 10 immediately prior to commencing treatment.Plasma is used for measuring drug concentrations, and cells are analyzedfor leukemia markers (eGFP or hCD4) and signaling biomarkers asdescribed herein.

This general assay known in the art may be used to demonstrate thateffective therapeutic doses of the compounds disclosed herein can beused for inhibiting the proliferation of leukemic cells.

Example 43: Cell Culture of Epithelial Cells of Ocular Origin

Ocular epithelial cells are obtained within 5 days postmortempost-mortem from corneas preserved under cold storage conditions inOptisol (Bausch and Lomb, Irvine, Calif.) or from corneal biopsy fromliving donors. The tissue is washed with phosphate-buffered saline andincubated in Dispase II (Roche Diagnostics, Basel, Switzerland) at 37°C. for 30 minutes, and the epithelial surface is gently scraped toseparate the epithelium from the underlying stroma. The separatedepithelium is then incubated and pipetted intrypsin-ethylenediaminetetraacetic acid to obtain a single cellsuspension. The trypsin is then neutralized with corneal epitheliumculture medium. Corneal epithelium culture medium is composed ofDulbecco modified Eagle medium:F12 basal media in a 2:1 ratio containing10% irradiated fetal bovine serum, hydrocortisone 0.4 μg/mL, choleratoxin 0.1 nmol, recombinant human insulin 5 μg/mL, and epidermal growthfactor 10 ng/mL, and the antimicrobials penicillin (100 IU/mL),streptomycin (100 μg/mL), and amphotericin B (0.25 μg/mL). Cells aremaintained by sub-culturing at a 1:4 ratio after reaching 80%confluency. Ocular epithelial cells are screened for inhibition ofproliferation or toxicity by contacting a test compound with the cellsand assaying for viability using the commercially available MTT assay(Promega).

Example 44: Cell Culture of Endothelial Cells of Ocular Origin

All tissues are maintained at 4° C. in storage medium (Optisol; ChironVision, Irvine, Calif.) for less than 10 days before study. The tissueis rinsed three times with DMEM containing 50 mg/mL gentamicin and 1.25mg/mL amphotericin B. The central cornea is removed by a trephine of8-mm diameter. Afterward, the Descemet's membrane and cornealendothelial cells are stripped from the posterior surface of theperipheral corneoscleral tissue under a dissecting microscope anddigested at 37° C. for 1.5 to 16 hours with 2 mg/mL collagenase A insupplemented hormonal epithelial medium (SHEM), which is made of anequal volume of HEPES-buffered DMEM and Ham's F12 supplemented with 5%FBS, 0.5% dimethyl sulfoxide, 2 ng/mL mouse EGF, 5 μg/mL insulin, 5μg/mL transferrin, 5 ng/mL selenium, 0.5 μg/mL hydrocortisone, 1 nMcholera toxin, 50 μg/mL gentamicin, and 1.25 μg/mL amphotericin B. Afterdigestion, HCECs formed aggregates, which are collected bycentrifugation at 2000 rpm for 3 minutes to remove the digestionsolution. As a control, Descemet's membrane strips are also digested in10 mg/mL Dispase II in SHEM and trypsin/EDTA for up to 3 hours.

Preservation of Isolated HCEC Aggregates

The resultant aggregates of HCECs are preserved in KSFM with completesupplement (storage medium 1), DMEM/F12 with KSFM supplements (storagemedium 2), or DMEM/F12 with SHEM supplements without FBS (storage medium3). All these media are serum free, one of the major differences amongthem is the calcium concentration, which is 0.09 mM in storage medium 1,but is 1.05 mM in storage media 2 and 3. HCEC aggregates are stored in atissue culture incubator at 37° C. for up to 3 weeks. Cell viability isdetermined (Live and Dead assay; Invitrogen) and also evaluated bysubculturing them in SHEM.

Expansion of Isolated HCEC Aggregates

The resultant HCEC aggregates, either immediately after digestion orafter a period of preservation in a storage medium, are then cultured inSHEM with or without additional growth factors such as 40 ng/mL bFGF,0.1 mg/mL BPE, and 20 ng/mL NGF on a plastic dish under 37° C. and 5%CO2. The media are changed every 2 to 3 days. Some HCEC aggregates arepretreated with trypsin/EDTA at 37° C. for 10 minutes to dissociateendothelial cells before the aforementioned cultivation.

Immunostaining

HCEC aggregates are embedded in OCT and subjected to frozen sectioning.Cryosections of 4 μm are air-dried at room temperature (RT) for 30minutes, and fixed in cold acetone for 10 minutes at −20° C. Sectionsused for immunostaining are rehydrated in PBS, and incubated in 0.2%Triton X-100 for 10 minutes. After three rinses with PBS for 5 minuteseach and preincubation with 2% BSA to block nonspecific staining, thesections are incubated with anti-laminin 5, type IV collagen, perlecan,ZO-1, and connexin 43 (all at 1:100) antibodies for 1 hour. After threewashes with PBS for 15 minutes, the sections are incubated with aFITC-conjugated secondary antibody (goat anti-rabbit or anti-mouse IgGat 1:100) for 45 minutes. After three additional PBS washes, each for 10minutes, they are counterstained with propidium iodide (1:1000) orHoechst 33342 (10 μg/mL), then mounted with an antifade solution andanalyzed with a fluorescence microscope. HCECs cultured in 24-wellplates or chamber slides are fixed in 4% paraformaldehyde for 15 minutesat RT and stained with anti-ZO-1 and connexin 43 antibodies as justdescribed. For immunohistochemical staining of Ki67, endogenousperoxidase activity is blocked by 0.6% hydrogen peroxide for 10 minutes.Nonspecific staining is blocked by 1% normal goat serum for 30 minutes.Cells are then incubated with anti-Ki67 antibody (1:100) for 1 hour.After three washes with PBS for 15 minutes, cells are incubated withbiotinylated rabbit anti-mouse IgG (1:100) for 30 minutes, followed byincubation with ABC reagent for 30 minutes. The reaction product isdeveloped with DAB for 5 minutes and examined by light microscope.

Cell-Viability and TUNEL Assays

Cell-viability and terminal deoxyribonucleotidyl transferase-mediatedFITC-linked dUTP nick-end DNA labeling (TUNEL) assays are used todetermine living and apoptotic cells, respectively. HCEC aggregates areincubated with cell-viability assay reagents for 15 minutes at RT. Livecells are distinguished by green fluorescence staining of the cellcytoplasm, and dead cells are stained with red fluorescence in thenuclei. The TUNEL assay is performed according to the manufacturer'sinstructions. Briefly, cross-sections of HCEC aggregates are fixed in 4%paraformaldehyde for 20 minutes at RT and permeabilized with 1% TritonX-100. Samples are then incubated for 60 minutes at 37° C. withexogenous TdT and fluorescein-conjugated dUTP, for repair of nicked3′-hydroxyl DNA ends. Cells are treated with DNase I as the positivecontrol, whereas negative control cells are incubated with a bufferlacking the rTdT enzyme. The apoptotic nuclei are labeled with greenfluorescence.

Example 45: Cell Culture of Retinal Cells

Eyes are cut in half along their equator and the neural retina isdissected from the anterior part of the eye in buffered saline solution,according to standard methods known in the art. Briefly, the retina,ciliary body, and vitreous are dissected away from the anterior half ofthe eye in one piece, and the retina is gently detached from the clearvitreous. Each retina is dissociated with papain (WorthingtonBiochemical Corporation, Lakewood, N.J.), followed by inactivation withfetal bovine serum (FBS) and addition of 134 Kunitz units/ml of DNaseI.The enzymatically dissociated cells are triturated and collected bycentrifugation, resuspended in Dulbecco's modified Eagle's medium(DMEM)/F12 medium (Gibco BRL, Invitrogen Life Technologies, Carlsbad,Calif.) containing 25 μg/ml of insulin, 100 μg/ml of transferrin, 60 pMputrescine, 30 nM selenium, 20 nM progesterone, 100 U/ml of penicillin,100 μg/ml of streptomycin, 0.05M Hepes, and 10% FBS. Dissociated primaryretina 1 cells are plated onto Poly-D-lysine- and Matrigel- (BD,Franklin Lakes, N.J.) coated glass coverslips that are placed in 24-welltissue culture plates (Falcon Tissue Culture Plates, Fisher Scientific,Pittsburgh, Pa.). Cells are maintained in culture for 5 days to onemonth in 0.5 ml of media (as above, except with only 1% FBS) at 37° C.and 5% CO2.

Immunocytochemistry Analysis

The retina 1 neuronal cells are cultured for 1, 3, 6, and 8 weeks in thepresence and absence of test compounds of the present invention, and thecells are analyzed by immunohistochemistry at each time point.Immunocytochemistry analysis is performed according to standardtechniques known in the art. Rod photoreceptors are identified bylabeling with a rhodopsin-specific antibody (mouse monoclonal, diluted1:500; Chemicon, Temecula, Calif.). An antibody to mid-weightneurofilament (NFM rabbit polyclonal, diluted 1:10,000, Chemicon) isused to identify ganglion cells; an antibody to β3-tubulin (G7121 mousemonoclonal, diluted 1:1000, Promega, Madison, Wis.) is used to generallyidentify interneurons and ganglion cells, and antibodies to calbindin(AB1778 rabbit polyclonal, diluted 1:250, Chemicon) and calretinin(AB5054 rabbit polyclonal, diluted 1:5000, Chemicon) are used toidentify subpopulations of calbindin- and calretinin-expressinginterneurons in the inner nuclear layer. Briefly, the retina 1 cellcultures are fixed with 4% paraformaldehyde (Polysciences, Inc,Warrington, Pa.) and/or ethanol, rinsed in Dulbecco's phosphate bufferedsaline (DPBS), and incubated with primary antibody for 1 hour at 37° C.The cells are then rinsed with DPBS, incubated with a secondary antibody(Alexa 488- or Alexa 568-conjugated secondary antibodies (MolecularProbes, Eugene, Oreg.)), and rinsed with DPBS. Nuclei are stained with4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes), and the culturesare rinsed with DPBS before removing the glass coverslips and mountingthem with Fluoromount-G (Southern Biotech, Birmingham, Ala.) on glassslides for viewing and analysis.

Example 46: Matrigel Plug Angiogenesis Assay

Matrigel containing test compounds are injected subcutaneously orintraocularly, where it solidifies to form a plug. The plug is recoveredafter 7-21 days in the animal and examined histologically to determinethe extent to which blood vessels have entered it. Angiogenesis ismeasured by quantification of the vessels in histologic sections.Alternatively, fluorescence measurement of plasma volume is performedusing fluorescein isothiocyanate (FITC)-labeled dextran 150. The resultsare expected to indicate one or more compounds disclosed herein thatinhibit angiogenesis and are thus expected to be useful in treatingocular disorders related to aberrant angiogenesis and/or vascularpermeability.

Example 47: The Corneal Angiogenesis Assay

A pocket is made in the cornea, and a plug containing an angiogenesisinducing formulation (e.g. VEGF, FGF, or tumor cells), when introducedinto this pocket, elicits the ingrowth of new vessels from theperipheral limbal vasculature. Slow-release materials such as ELVAX(ethylene vinyl copolymer) or Hydron are used to introduce angiogenesisinducing substances into the corneal pocket. Alternatively, a spongematerial is used.

The effect of putative inhibitors on the locally induced (e.g., spongeimplant) angiogenic reaction in the cornea (e.g., by FGF, VEGF, or tumorcells). The test compound is administered orally, systemically, ordirectly to the eye. Systemic administration is by bolus injection or,more effectively, by use of a sustained-release method such asimplantation of osmotic pumps loaded with the test inhibitor.Administration to the eye is by any of the methods described hereinincluding but not limited to eye drops, topical administration of acream, emulsion, or gel, intravitreal injection.

The vascular response is monitored by direct observation throughout thecourse of the experiment using a stereomicroscope in mice. Definitivevisualization of the corneal vasculature is achieved by administrationof fluorochrome-labeled high-molecular weight dextran. Quantification isperformed by measuring the area of vessel penetration, the progress ofvessels toward the angiogenic stimulus over time, or in the case offluorescence, histogram analysis or pixel counts above a specific(background) threshold.

The results are expected to indicate one or more compounds disclosedherein that inhibit angiogenesis and are thus expected to be useful intreating ocular disorders related to aberrant angiogenesis and/orvascular permeability.

Example 48: Microtiter-Plate Angiogenesis Assay

The assay plate is prepared by placing a collagen plug in the bottom ofeach well with 5-10 cell spheroids per collagen plug each spheroidcontaining 400-500 cells. Each collagen plug is covered with 1100 μl ofstorage medium per well and stored for future use (1-3 days at 37° C.,5% CO2). The plate is sealed with sealing. Test compounds are dissolvedin 200 μl assay medium with at least one well including a VEGF positivecontrol and at least one well without VEGF or test compound as anegative control. The assay plate is removed from the incubator andstorage medium is carefully pipeted away. Assay medium containing thetest compounds are pipeted onto the collagen plug. The plug is placed ina humidified incubator for (37° C., 5% CO2) 24-48 hours. Angiogenesis isquantified by counting the number of sprouts, measuring average sproutlength, or determining cumulative sprout length. The assay can bepreserved for later analysis by removing the assay medium, adding 1 mlof 10% paraformaldehyde in Hanks BSS per well, and storing at 4° C. Theresults are expected to identify compounds that inhibit angiogenesis invarious cell types tested, including cells of ocular origin.

Example 49: TNP-Ficoll T-Cell Independent B-Cell Activation Assay

To test the effects of the compounds of the present invention insuppressing T cell independent antibody production, the TNP-FicollB-cell activation assay was used as described herein. Compounds of thepresent invention were dissolved in an appropriate vehicle (e.g. 5%1-methyl-2-pyrrolidinone, 85% polyethylene glycol 400, 10% solutor).Compounds were administered orally approximately 1 hr before TNP-Ficolltreatment to 4-10 week old mice. To study the effects of the compoundson B-cell activation, one set of mice were grouped according to thefollowing table:

Antigen injection Compound Administration Mice/ Comp at day-1 from day-1to day-7 Group# group treated Group TNP-F Route (mg/kg) Route Regimen 14 Vehicle Antigen only 200 uL ip 0 Po BID for 7 2 8 — Antigen only (0.5mg/ml) 0 days 3 8 Compound reference 30 #7 4 8 Compound Antigen + cmp 15 8 #53 3 6 8 10 7 8 30 8 8 60

Four animals in group 1, and eight animals in groups 2 to 7 wereeuthanized in CO2 2 hours after the last compound administration on day7. Blood was immediately collected by cadio-puncture and kept at 37° C.for 1 hr to clot followed by overnight incubation at 4° C. to allow theclot to contract. The following day, serum was collected by decantingand centrifugation at 3000 rpm for 10 min. The collected serum was thenfrozen at −80° C. for future analysis.

Serum samples were analyzed for anti-TNP antibody titers by ELISA asdescribed herein. TNP-BSA was coated onto a Nunc Maxisorb microtiterplate with 100 μl/well at a concentration of 10 g/ml in phosphatebuffered saline (PBS). The Maxisorb plate was incubated for 1.5 hours atroom temperature and the solution was removed. 200 μl/well of blockingbuffer (e.g. 1% BSA in PBS) was added to each well and incubated 1 hr atroom temperature. The plate was washed once with 200 μl/well of PBS0.05% Tween-20 (wash buffer). A 1:2 dilution of serum from each mouse inblocking buffer was added to each well in the first column (1) of themicrotiter plate. The serum in each well of column 1 was then diluted3-fold in blocking buffer and added to column 2. The serum in each wellof column 2 was diluted 3-fold in blocking buffer and added to column 3.The procedure was repeated across the twelve columns of the microtiterplate. The microtiter plate was incubated 1 hr at room temperature.Serum was removed from the plate and the plate was washed three timeswith wash buffer. 100 μl/well of goat anti-mouse IgG3-HRP diluted 1:250in blocking buffer was added to each well and incubated 1 hr at roomtemperature. The anti-mouse IgG3-HRP was removed from the microtiterplate and the plate was washed six times with wash buffer. HRP substrate(200 μl ABTS solution+30% H2O2+10 ml citrate buffer) was added to eachwell at 100 μl/well, incubated 2-20 minutes in the dark and the amountof anti-TNP IgG3 was determined spectrophotometrically at 405 nm.Similarly, anti-TNP IgM and total anti-TNP Ab were determined usinganti-mouse IgM-HRP and anti-mouse Ig-HRP respectively.

The results as shown in FIG. 2 further show that under the conditionstested compounds #7 and #53 exhibit 3.4 and 6.5-fold reductionsrespectively in IgG3 levels relative to vehicle control mice at a 30mg/kg dose level. FIG. 2 further shows that compound #53 exhibits29.9-fold reduction in IgG3 levels relative to vehicle control mice at a60 mg/kg dose level under the conditions tested,

Example 50: Rat Developing Type II Collagen Induced Arthritis Assay

In order to study the effects of the compounds of the present inventionon the autoimmune disease arthritis, a collagen induced developingarthritis model was used. Female Lewis rats were given collageninjections at day 0. Bovine type II collagen was prepared as a 4 mg/mlsolution in 0.01N acetic acid. Equal volumes of collagen and Freund'sincomplete adjuvant were emulsified by hand mixing until a bead of theemulsified material held its form in water. Each rodent received a 300μl injection of the mixture at each injection time spread over threesubcutaneous sites on the back.

Oral compound administration began on day 0 and continued through day 16with vehicle (5% NMP, 85% PEG 400, 10% Solutol) or compounds of thepresent invention in vehicle or control (e.g. methotrexate) at 12 hourintervals daily. Rats were weighed on days 0, 3, 6, 9-17 and calipermeasurements of ankles taken on days 9-17. Final body weights weretaken, and then the animals were euthanized on day 17. Aftereuthanization, blood was drawn and hind paws and knees were removed.Blood was further processed for pharmacokinetics experiments as well asan anti-type II collagen antibody ELISA assay. Hind paws were weighedand then with the knees preserved in 10% formalin. The paws and kneeswere subsequently processed for microcopy. Livers, spleen and thymuswere also weighed. Sciatic nerves were prepared for histopathology.

Knee and ankle joints were fixed for 1-2 days and decalcified for 4-5days. Ankle joints were cut in half longitudinally, knees were cut inhalf along the frontal plane. Joints were then processed, embedded,sectioned and stained with toluidine blue. Scoring of the joints wasdone according to the following criteria:

Knee and Ankle Inflammation

-   -   0=Normal    -   1=Minimal infiltration of inflammatory cells in        synovium/periarticular tissue    -   2=Mild infiltration    -   3=Moderate infiltration with moderate edema    -   4=Marked infiltration with marked edema    -   5=Severe infiltration with severe edema

Ankle Pannus

-   -   0=Normal    -   1=Minimal infiltration of pannus in cartilage and subchondral        bone    -   2=Mild infiltration (<¼ of tibia or tarsals at marginal zones)    -   3=Moderate infiltration (¼ to ⅓ of tibia or small tarsals        affected at marginal zones)    -   4=Marked infiltration (½-¾ of tibia or tarsals affected at        marginal zones)    -   5=Severe infiltration (>¾ of tibia or tarsals affected at        marginal zones, severe distortion of overall architecture)

Knee Pannus

-   -   0=Normal    -   1=Minimal infiltration of pannus in cartilage and subchondral        bone    -   2=Mild infiltration (extends over up to ¼ of surface or        subchondral area of tibia or femur)    -   3=Moderate infiltration (extends over >¼ but <½ of surface or        subchondral area of tibia or femur)    -   4=Marked infiltration (extends over ½ to ¾ of tibial or femoral        surface)    -   5=Severe infiltration (covers >¾ of surface)

Cartilage Damage (Ankle, Emphasis on Small Tarsals)

-   -   0=Normal    -   1=Minimal=minimal to mild loss of toluidine blue staining with        no obvious chondrocyte loss or collagen disruption    -   2=Mild=mild loss of toluidine blue staining with focal mild        (superficial) chondrocyte loss and/or collagen disruption    -   3=Moderate=moderate loss of toluidine blue staining with        multifocal moderate (depth to middle zone) chondrocyte loss        and/or collagen disruption, smaller tarsals affected to ½-¾        depth    -   4=Marked=marked loss of toluidine blue staining with multifocal        marked (depth to deep zone) chondrocyte loss and/or collagen        disruption, 1 or more small tarsals have full thickness loss of        cartilage    -   5=Severe=severe diffuse loss of toluidine blue staining with        multifocal severe (depth to tide mark) chondrocyte loss and/or        collagen disruption

Cartilage Damage (Knee, Emphasis on Femoral Condyles)

-   -   0=Normal    -   1=Minimal=minimal to mild loss of toluidine blue staining with        no obvious chondrocyte loss or collagen disruption    -   2=Mild=mild loss of toluidine blue staining with focal mild        (superficial) chondrocyte loss and/or collagen disruption    -   3=Moderate=moderate loss of toluidine blue staining with        multifocal to diffuse moderate (depth to middle zone)        chondrocyte loss and/or collagen disruption    -   4=Marked=marked loss of toluidine blue staining with multifocal        to diffuse marked (depth to deep zone) chondrocyte loss and/or        collagen disruption or single femoral surface with total or near        total loss    -   5=Severe=severe diffuse loss of toluidine blue staining with        multifocal severe (depth to tide mark) chondrocyte loss and/or        collagen disruption on both femurs and/or tibias

Bone Resorption (Ankle)

-   -   0=Normal    -   1=Minimal=small areas of resorption, not readily apparent on low        magnification, rare osteoclasts    -   2=Mild=more numerous areas of resorption, not readily apparent        on low magnification, osteoclasts more numerous, <¼ of tibia or        tarsals at marginal zones resorbed    -   3=Moderate=obvious resorption of medullary trabecular and        cortical bone without full thickness defects in cortex, loss of        some medullary trabeculae, lesion apparent on low magnification,        osteoclasts more numerous, ¼ to ⅓ of tibia or tarsals affected        at marginal zones    -   4=Marked=Full thickness defects in cortical bone, often with        distortion of profile of remaining cortical surface, marked loss        of medullary bone, numerous osteoclasts, ½-¾ of tibia or tarsals        affected at marginal zones    -   5=Severe=Full thickness defects in cortical bone, often with        distortion of profile of remaining cortical surface, marked loss        of medullary bone, numerous osteoclasts, >¾ of tibia or tarsals        affected at marginal zones, severe distortion of overall        architecture

Bone Resorption (Knee)

-   -   0=Normal    -   1=Minimal=small areas of resorption, not readily apparent on low        magnification, rare osteoclasts    -   2=Mild=more numerous areas of resorption, definite loss of        subchondral bone involving ¼ of tibial or femoral surface        (medial or lateral)    -   3=Moderate=obvious resorption of subchondral bone involving >¼        but <½ of tibial or femoral surface (medial or lateral)    -   4=Marked=obvious resorption of subchondral bone involving >½ but        <¾ of tibial or femoral surface (medial or lateral)    -   5=Severe=distortion of entire joint due to destruction        involving >¾ of tibial or femoral surface (medial or lateral)

Statistical analysis of body/paw weights, paw AUC parameters andhistopathologic parameters were evaluated using a Student's t-test orother appropriate (ANOVA with post-test) with significance set at the 5%significance level. Percent inhibition of paw weight and AUC wascalculated using the following formula:

% Inhibition=A−B/A×100

A=Mean Disease Control−Mean Normal

B=Mean Treated−Mean Normal

The results as shown in FIG. 3 demonstrate the effect of compound #53 at10, 30, and 60 mg/kg dosages at 12 hour intervals on mean ankle diameterover time in a rat developing type II collagen induced arthritis modelunder the conditions tested. Relative to the vehicle alone control or tothe methotrexate control, the compounds of the present inventionexhibited a significant reduction in arthritis induced ankle diameterincrease over time.

The results as shown in FIG. 4 demonstrate the effect of compounds #7and #53 on ankle histopathology in the categories of inflammation,pannus, cartilage damage, and bone resorption as previously describedunder the conditions tested. The results show a significant reduction inone or more categories by one of the compounds of the present invention(i.e. compound #53) under the conditions tested. FIG. 4 further showsthat at 60 mg/kg, there is a statistically significant reduction in allcategories of ankle histopathology for one of the compounds of thepresent invention (i.e. compound #53) under the conditions tested. Thissuggests that one or more compounds of the present invention may beuseful for the treatment and reduction of arthritis disease symptoms.

The results as shown in FIG. 5 demonstrate the effect of compounds #7and #53 on knee histopathology under the conditions tested. The resultsdemonstrate a dose dependent reduction in knee histopathology. Thissuggests that one or more compounds of the present invention may beuseful for the treatment and reduction of arthritis disease symptoms.

The results as shown in FIG. 6 demonstrate the effect of the compounds#7 and #53 on serum anti-type II collagen levels under the conditionstested. The results further show a significant reduction at 10, 20, and60 mg/kg dosage levels of serum anti-type II collagen levels forcompound #53, suggesting that one or more compounds of the presentinvention may not only be useful for the treatment and reduction ofarthritis disease symptoms, but may also be useful for the inhibition ofthe autoimmune reaction itself.

The results as shown in FIG. 7 demonstrate the effect of compound #7 at10, 30, and 60 mg/kg dosages at 12 hour intervals on mean ankle diameterover time under the conditions tested. Relative to the vehicle alonecontrol or to the methotrexate control, the compound exhibited areduction in arthritis induced ankle diameter increase over time underthe conditions tested.

Example 51: Rat Established Type II Collagen Induced Arthritis Assay

In order to examine the dose responsive efficacy of the compounds of thepresent invention in inhibiting the inflammation, cartilage destructionand bone resorption of 7 day established type II collagen inducedarthritis in rats, compounds were administered orally daily or twicedaily for 6 days.

Female Lewis rats were anesthetized and given collagen injectionsprepared and administered as described previously on day 0. On day 6,animals were anesthetized and given a second collagen injection. Calipermeasurements of normal (pre-disease) right and left ankle joints wereperformed on day 9. On days 10-11, arthritis typically occurred and ratswere randomized into treatment groups. Randomization was performed afterankle joint swelling was obviously established and there was goodevidence of bilateral disease.

After an animal was selected for enrollment in the study, treatment wasinitiated by the oral route. Animals were given vehicle, control(Enbrel) or compound doses, twice daily or once daily (BID or QDrespectively). Dosing was administered on days 1-6 using a volume of 2.5ml/kg (BID) or 5 ml/kg (QD) for oral solutions. Rats were weighed ondays 1-7 following establishment of arthritis and caliper measurementsof ankles taken every day. Final body weights were taken on day 7 andanimals were euthanized.

The results as shown in FIG. 8 shows a significant reduction in meanankle diameter increase over time for compound #53 with a once dailydosage under the conditions tested. The results in FIG. 9 furtherdemonstrate a significant reduction in mean ankle diameter increase overtime for compound #53 with a twice daily dosage under the conditionstested. This suggests that the compounds of the present invention may beuseful for the treatment of autoimmune diseases such as arthritis.

Example 52: Adjuvant Induced Arthritis Assay Intrathecal Catheterizationof Rats

Isoflurane-anesthetized Lewis rats (200-250 g) were implanted with anintrathecal (IT) catheter. After a 6 d recovery period, all animalsexcept those that appeared to have sensory or motor abnormalities (fewerthan 5% of the total number) were used for experiments. For ITadministration, 10 μl of drug or saline followed by 10 μl of isotonicsaline was injected through the catheter.

Adjuvant Arthritis and Drug Treatment

Lewis rats were immunized at the base of the tail with 0.1 ml ofcomplete Freund's adjuvant (CFA) on day 0 several days after catheterimplantation (n=6/group). Drug (e.g. one or more compounds of thepresent invention or or vehicle) treatment was generally started on day8 and continued daily until day 20. Clinical signs of arthritisgenerally begin on day 10, and paw swelling was determined every secondday by water displacement plethysmometry.

The results as depicted in FIG. 10 by the average change in paw volumeunder the dosage regimes indicated show that under the conditionstested, compound #53 shows a dose dependent reduction in the average pawvolume increase as measured in this adjuvant induced arthritis modelsystem. These results suggest that one or more of the compounds of thepresent invention may be useful for the treatment of one or more of thediseases or conditions described herein.

The results as depicted in FIG. 11 show that compound #53 does notexhibit toxicity or other adverse reaction under the conditions testedas measured by a lack of weight loss.

Example 53: Rodent Pharmacokinetic Assay

In order to study the pharmacokinetics of the compounds of the presentinvention a set of 4-10 week old mice are grouped according to thefollowing table:

Compound Administration Mice/ Compound from day-1 to day-7 Group# grouptreated (mg/kg) Route Regimen 1 3 1 Po BID for 7 2 3 3 days 3 3 10 4 330 5 3 60

Compounds of the present invention are dissolved in an appropriatevehicle (e.g. 5% 1-methyl-2-pyrrolidinone, 85% polyethylene glycol 400,10% Solutor) and administered orally at 12 hour intervals daily. Allanimals are euthanized in CO2 2 hours after the final compound isadministered. Blood is collected immediately and kept on ice for plasmaisolation. Plasma is isolated by centrifuging at 5000 rpm for 10minutes. Harvested plasma is frozen for pharmacokinetic detection.

The results are expected to demonstrate the pharmacokinetic parameterssuch as absorption, distribution, metabolism, excretion, and toxicityfor the compounds of the present invention.

Example 54: Basotest Assay

The baseotest assay is performed using Orpegen Pharma Basotest reagentkit. Heparinized whole blood is pre-incubated with test compound orsolvent at 37 C for 20 min. Blood is then incubated with assay kitstimulation buffer (to prime cells for response) followed by allergen(dust mite extract or grass extract) for 20 min. The degranulationprocess is stopped by incubating the blood samples on ice. The cells arethen labeled with anti-IgE-PE to detect basophilic granulocytes, andanti-gp53-FITC to detect gp53 (a glycoprotein expressed on activatedbasophils). After staining red blood cells are lysed by addition ofLysing Solution. Cells are washed, and analyzed by flow cytometry.Compounds 7 and 53 when tested in this assay inhibit allergen inducedactivation of basophilic granulocytes at sub micromolar range.

Example 55: Combination Use of PI3Kδ Inhibitors and Agents that InhibitIgE Production or Activity

The compounds of the present invention may present synergistic oradditive efficacy when administered in combination with agents thatinhibit IgE production or activity. Agents that inhibit IgE productioninclude, for example, one or more of TEI-9874,2-(4-(6-cyclohexyloxy-2-naphtyloxy)phenylacetamide)benzoic acid,rapamycin, rapamycin analogs (i.e. rapalogs), TORC1 inhibitors, TORC2inhibitors, and any other compounds that inhibit mTORC1 and mTORC2.Agents that inhibit IgE activity include, for example, anti-IgEantibodies such as Omalizumab and TNX-901.

One or more of the subject compounds capable of inhibiting PI3Kδ areefficacious in treatment of autoimmune and inflammatory disorders (AIID)for example rheumatoid arthritis. If any of the compounds causes anundesired level of IgE production, one may choose to administer it incombination with an agent that inhibits IgE production or IgE activity.Additionally, the administration of PI3Kδ or PI3Kδ/γ inhibitors of thepresent invention in combination with inhibitors of mTOR may alsoexhibit synergy through enhanced inhibition of the PI3K pathway. Variousin vivo and in vitro models may be used to establish the effect of suchcombination treatment on AIID including but not limited to (a) in vitroB-cell antibody production assay, (b) in vivo TNP assay, and (c) rodentcollagen induced arthritis model.

(a) B-Cell Assay

Mice are euthanized, and the spleens are removed and dispersed through anylon mesh to generate a single-cell suspension. The splenocytes arewashed (following removal of erythrocytes by osmotic shock) andincubated with anti-CD43 and anti-Mac-1 antibody-conjugated microbeads(Miltenyi Biotec). The bead-bound cells are separated from unbound cellsusing a magnetic cell sorter. The magnetized column retains the unwantedcells and the resting B cells are collected in the flow-through.Purified B-cells are stimulated with lipopolysaccharide or an anti-CD40antibody and interleukin 4. Stimulated B-cells are treated with vehiclealone or with PI3Kδ inhibitors of the present invention such as compound53 with and without mTOR inhibitors such as rapamycin, rapalogs, ormTORC1/C2 inhibitors. The results are expected to show that in thepresence of mTOR inhibitors (e.g., rapamycin) alone, there is little tono substantial effect on IgG and IgE response. However, in the presenceof PI3Kδ and mTOR inhibitors, the B-cells are expected to exhibit adecreased IgG response as compared to the B-cells treated with vehiclealone, and the B-cells are expected to exhibit a decreased IgE responseas compared to the response from B-cells treated with PI3Kδ inhibitorsalone.

(b) TNP Assay

Mice are immunized with TNP-Ficoll or TNP-KHL and treated with: vehicle,a PI3Kδ inhibitor, for example, compound 53 of the present invention, anmTOR inhibitor, for example rapamycin, or a PI3Kδ inhibitor incombination with an mTOR inhibitor such as rapamycin. Antigen-specificserum IgE is measured by ELISA using TNP-BSA coated plates and isotypespecific labeled antibodies. It is expected that mice treated with anmTOR inhibitor alone exhibit little or no substantial effect on antigenspecific IgG3 response and no statistically significant elevation in IgEresponse as compared to the vehicle control. It is also expected thatmice treated with both PI3Kδ inhibitor and mTOR inhibitor exhibit areduction in antigen specific IgG3 response as compared to the micetreated with vehicle alone. Additionally, the mice treated with bothPI3Kδ inhibitor and mTOR inhibitor exhibit a decrease in IgE response ascompared to the mice treated with PI3Kδ inhibitor alone.

(c) Rat Collagen Induced Arthritis Model

Female Lewis rats are anesthetized and given collagen injectionsprepared and administered as described previously on day 0. On day 6,animals are anesthetized and given a second collagen injection. Calipermeasurements of normal (pre-disease) right and left ankle joints areperformed on day 9. On days 10-11, arthritis typically occurs and ratsare randomized into treatment groups. Randomization is performed afterankle joint swelling is obviously established and there is good evidenceof bilateral disease.

After an animal is selected for enrollment in the study, treatment isinitiated. Animals are given vehicle, PI3Kδ inhibitor, or PI3Kδinhibitor in combination with rapamycin. Dosing is administered on days1-6. Rats are weighed on days 1-7 following establishment of arthritisand caliper measurements of ankles taken every day. Final body weightsare taken on day 7 and animals are euthanized.

It is expected that the combination treatment using PI3Kδ inhibitor andrapamycin provides greater efficacy than treatment with PI3Kδ inhibitoralone.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1-59. (canceled)
 60. A compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: W_(a) ¹ is CR³;W_(a) ² is CR⁵; W_(a) ³ is CR⁶; W_(a) ⁴ is N; W_(b) ⁵ is CR⁸ or CHR⁸;W_(d) is

R^(a) is hydrogen, halo, phosphate, urea, carbonate, alkyl, alkenyl,alkynyl, cycloalkyl, heteroalkyl, or heterocycloalkyl; R¹¹ is hydrogen,alkyl, halo, amino, amido, hydroxy, or alkoxy; R¹² is hydrogen, alkyl,cyano, alkynyl, alkenyl, halo, aryl, heteroaryl, heterocycloalkyl,cycloalkyl, amino, carboxylic acid, alkoxycarbonyl, or amido; B isalkyl, amino, heteroalkyl, cycloalkyl, heterocycloalkyl, or a moiety ofFormula II:

W_(c) is aryl, heteroaryl, heterocycloalkyl, or cycloalkyl; q is aninteger of 0, 1, 2, 3, or 4; X is absent or is —(CH(R⁹))_(z)— and eachinstance of z is independently an integer of 1, 2, 3, or 4; Y is absent,—O—, —S—, —S(═O)—, —S(═O)₂—, —N(R⁹)—, —C(═O)(CHR⁹)_(z)—, —C(═O)—,—N(R⁹)C(═O)NH—, or —N(R⁹)C(R⁹)₂—, wherein when W_(b) ⁵ is N, no morethan one of X or Y is absent; R¹ is hydrogen, alkyl, heteroalkyl,alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl,heteroaryl, heteroarylalkyl, alkoxy, amido, amino, acyl, acyloxy,alkoxycarbonyl, sulfonamido, halo, cyano, hydroxy, nitro, phosphate,urea, or carbonate; R² is alkyl, heteroalkyl, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl,sulfonamido, halo, cyano, hydroxy or nitro; R³ is hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkoxy, amido, amino,acyl, acyloxy, alkoxycarbonyl, sulfonamide, halo, cyano, hydroxyl,nitro, aryl, or heteroaryl; R⁵, R⁶, and R⁸ are independently hydrogen,C₁-C₄alkyl, C₂-C₅alkenyl, C₂-C₅alkynyl, C₃-C₅cycloalkyl,C₁-C₄heteroalkyl, C₁-C₄alkoxy, C₁-C₄amido, amino, acyl, C₁-C₄acyloxy,C₁-C₄sulfonamido, halo, cyano, hydroxy or nitro; and each instance of R⁹is independently hydrogen, C₁-C₁₀alkyl, C₃-C₇cycloalkyl, orC₂-C₁₀heteroalkyl.
 61. The compound of claim 60, wherein the compound isa compound of Formula VII:

or a pharmaceutically acceptable salt thereof.
 62. The compound of claim60, wherein W_(c) is aryl or heterocycloalkyl.
 63. The compound of claim60, wherein R³ is alkyl.
 64. The compound of claim 60, wherein R⁵, R⁶,and R⁸ are hydrogen.
 65. The compound of claim 60, wherein X is—(CH(R⁹))_(z)— and z is an integer of
 1. 66. The compound of claim 60,wherein Y is absent.
 67. The compound of claim 60, wherein R¹² is iodo,bromo,


68. The compound of claim 60, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 69. The compound of claim60, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 70. A compound of FormulaIX:

or a pharmaceutically acceptable salt thereof, wherein W_(a) ¹ and W_(a)² are independently CR⁵, S, N, or NR⁴; W_(a) ⁴ is independently CR⁷, S,N, or NR⁴; wherein no more than two adjacent ring atoms are nitrogen orsulfur, and when W_(a) ¹ is S, one of W_(a) ² and W_(a) ⁴ is N or NR⁴;W_(b) ⁵ is CR⁸, N, or NR⁸; B is alkyl, amino, heteroalkyl, cycloalkyl,heterocycloalkyl, or a moiety of Formula II;

W_(c) is aryl, heteroaryl, heterocycloalkyl, or cycloalkyl; q is aninteger of 0, 1, 2, 3, or 4; W_(d) is a heterocycloalkyl, aryl orheteroaryl moiety; X is —(CH(R⁹))_(z)—; each instance of z independentlyis an integer of 1; Y is absent; R¹ is hydrogen, alkyl, heteroalkyl,alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl,heteroaryl, heteroarylalkyl, alkoxy, amido, amino, acyl, acyloxy,alkoxycarbonyl, sulfonamido, halo, cyano, hydroxy or nitro; R² is alkyl,heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,arylalkyl, heteroaryl, heteroarylalkyl, alkoxy, amido, amino, acyl,acyloxy, alkoxycarbonyl, sulfonamido, halo, cyano, hydroxy, nitro,phosphate, urea, or carbonate; R⁴ is hydrogen, acyl, C₁-C₄alkyl,C₂-C₅alkenyl, C₂-C₅alkynyl, C₃-C₅cycloalkyl, or C₁-C₄heteroalkyl; R⁵,R⁷, and R⁸ are independently hydrogen, C₁-C₄alkyl, C₂-C₅alkenyl,C₂-C₅alkynyl, C₃-C₅cycloalkyl, C₁-C₄heteroalkyl, acyl, C₁-C₄alkoxy,C₁-C₄amido, amino, C₁-C₄acyloxy, C₁-C₄sulfonamido, halo, cyano, hydroxyor nitro; and each instance of R⁹ is independently hydrogen,C₁-C₁₀alkyl, C₃-C₇cycloalkyl, or C₂-C₁₀heteroalkyl.
 71. The compound ofclaim 70, wherein the compound is a compound of Formula XIII or FormulaX:

or a pharmaceutically acceptable salt thereof, wherein B is a moiety ofFormula II;

wherein W_(c) is aryl, heteroaryl, heterocycloalkyl, or cycloalkyl, andq is an integer of 0, 1, or 2; W_(d) is:

X is (CH₂)_(z); Y is absent; z is 1; R¹ is hydrogen, fluoro, chloro,cyano, methyl, isopropyl, —CF₃, methoxy, or nitro; R² is halo, hydroxy,cyano, or nitro; R⁴ is hydrogen or methyl; R⁵ is hydrogen or methyl; R⁷is hydrogen or methyl; R¹¹ is amino; and R¹² is hydrogen, alkyl,alkynyl, alkenyl, halo, aryl, heteroaryl, heterocycloalkyl, orcycloalkyl.
 72. The compound of claim 70, wherein R¹² is iodo, bromo,


73. The compound of claim 70, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 74. A pharmaceuticalcomposition comprising a compound of claim 60, or a pharmaceuticallyacceptable salt thereof, and one or more pharmaceutically acceptableexcipients.
 75. A pharmaceutical composition comprising a compound ofclaim 70, or a pharmaceutically acceptable salt thereof, and one or morepharmaceutically acceptable excipients.
 76. A method of treating adisorder comprising administering an effective amount of a compound ofclaim 60 to a subject suffering from the disorder, wherein the disorderis selected from cancer, bone disorder, inflammatory disease, immunedisease, nervous system disease, metabolic disease, respiratory disease,thrombosis, and cardiac disease.
 77. A method of treating a disordercomprising administering an effective amount of a compound of claim 70to a subject suffering from the disorder, wherein the disorder isselected from cancer, bone disorder, inflammatory disease, immunedisease, nervous system disease, metabolic disease, respiratory disease,thrombosis, and cardiac disease.